The genetic polymorphism of merozoite surface protein-1 in Plasmodium falciparum isolates from Aceh province, Indonesia

NASA Astrophysics Data System (ADS)

Jamil, K. F.; Supargiyono, S.; Syafruddin, D.; Pratama, N.; Silvy, S.

2018-03-01

An estimated of 3.3 million Indonesian population were infected with malaria. However, extensive genetic polymorphism of the field isolates msp-1 of P. falciparum represents a major obstacle for the development of malaria treatment. The aim of this study was to investigate the genetic diversity of msp-1 genotype in field isolates of P. falciparum collected in Aceh Province. A total of 90 patients with malaria (+) were selected from eleven district hospitals in Aceh from 2013-2015. Data were collected by anamnesis, complete physical examination and laboratory tests for msp-1. All protocols to diagnose malaria followed the WHO 2010 guideline. All samples were stored in Eijkman Biology Molecular Institute, Jakarta. Among 90 samples, 57.7% were male, and 42.3% were female with the most cases found between 21-30 years old. From the allele typing analysis of P. falciparum from Aceh; K1, MAD20, and RO33 allele types were identified. MAD20 type was the highest allele found in this study (57.9%). It was found in single and mixed infection. A moderate level of the mixed allele was also observed.

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

PubMed Central

Gentz, R; Certa, U; Takacs, B; Matile, H; Döbeli, H; Pink, R; Mackay, M; Bone, N; Scaife, J G

1988-01-01

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene. Images PMID:2452082

Genetic diversity of the DBLalpha region in Plasmodium falciparum var genes among Asia-Pacific isolates.

PubMed

Fowler, Elizabeth V; Peters, Jennifer M; Gatton, Michelle L; Chen, Nanhua; Cheng, Qin

2002-03-01

In Plasmodium falciparum a highly polymorphic multi-copy gene family, var, encodes the variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1), which has an important role in cytoadherence and immune evasion. Using previously described universal PCR primers for the first Duffy binding-like domain (DBLalpha) of var we analysed the DBLalpha repertoires of Dd2 (originally from Thailand) and eight isolates from the Solomon Islands (n=4), Philippines (n=2), Papua New Guinea (n=1) and Africa (n=1). We found 15-32 unique DBLalpha sequence types among these isolates and estimated detectable DBLalpha repertoire sizes ranging from 33-38 to 52-57 copies per genome. Our data suggest that var gene repertoires generally consist of 40-50 copies per genome. Eighteen DBLalpha sequences appeared in more than one Asia-Pacific isolate with the number of sequences shared between any two isolates ranging from 0 to 6 (mean=2.0 +/-1.6). At the amino acid level DBLalpha sequence similarity within isolates ranged from 45.2 +/- 7.1 to 50.2 +/- 6.9%, and was not significantly different from the DBLalpha amino acid sequence similarity among isolates (P>0.1). Comparisons with published sequences also revealed little overlap among DBLalpha sequences from different regions. High DBLalpha sequence diversity and minimal overlap among these isolates suggest that the global var gene repertoire is immense, and may potentially be selected for by the host's protective immune response to the var gene products, PfEMP1.

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

Artemisinin resistance without pfkelch13 mutations in Plasmodium falciparum isolates from Cambodia.

PubMed

Mukherjee, Angana; Bopp, Selina; Magistrado, Pamela; Wong, Wesley; Daniels, Rachel; Demas, Allison; Schaffner, Stephen; Amaratunga, Chanaki; Lim, Pharath; Dhorda, Mehul; Miotto, Olivo; Woodrow, Charles; Ashley, Elizabeth A; Dondorp, Arjen M; White, Nicholas J; Wirth, Dyann; Fairhurst, Rick; Volkman, Sarah K

2017-05-12

Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin-piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance. Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA 0-3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates. Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA 0-3h  = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance. This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine

Genetic diversity of Plasmodium falciparum isolates from naturally infected children in north-central Nigeria using the merozoite surface protein-2 as molecular marker.

PubMed

Oyedeji, Segun Isaac; Awobode, Henrietta Oluwatoyin; Anumudu, Chiaka; Kun, Jürgen

2013-08-01

To characterize the genetic diversity of Plasmodium falciparum (P. falciparum) field isolates in children from Lafia, North-central Nigeria, using the highly polymorphic P. falciparum merozoite surface protein 2 (MSP-2) gene as molecular marker. Three hundred and twenty children were enrolled into the study between 2005 and 2006. These included 140 children who presented with uncomplicated malaria at the Dalhatu Araf Specialist Hospital, Lafia and another 180 children from the study area with asymptomatic infection. DNA was extracted from blood spot on filter paper and MSP-2 genes were genotyped using allele-specific nested PCR in order to analyze the genetic diversity of parasite isolates. A total of 31 and 34 distinct MSP-2 alleles were identified in the asymptomatic and uncomplicated malaria groups respectively. No difference was found between the multiplicity of infection in the asymptomatic group and that of the uncomplicated malaria group (P>0.05). However, isolates of the FC27 allele type were dominant in the asymptomatic group whereas isolates of the 3D7 allele type were dominant in the uncomplicated malaria group. This study showed a high genetic diversity of P. falciparum isolates in North-central Nigeria and is comparable to reports from similar areas with high malaria transmission intensity. Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

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

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.

The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity.

PubMed

Delhaes, L; Lazaro, J E; Gay, F; Thellier, M; Danis, M

1999-01-01

Malarial lactate dehydrogenase (LDH), which uses 3-acetyl pyridine adenine dinucleotide as coenzyme in a reaction leading to the formation of pyruvate from L-lactate, may be used to study the susceptibility of Plasmodium falciparum to a drug in vitro. Several methods to determine the activity of this enzyme are available. One, the colorimetric method of Makler and colleagues, was modified slightly, by using sodium-2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5 - carboxanilide (XTT) and following the reaction by measuring the optical density at 450 nm. Using two, culture-adapted strains of P. falciparum, this LDH assay was compared with the unmodified Makler's assay and with the isotopic microtest based on the incorporation of tritium-labelled hypoxanthine. Fresh, clinical P. falciparum isolates were also tested in the presence of several drugs, including chloroquine, mefloquine, quinine, halofantrine, atovaquone and qinghaosu derivatives. The results of the three assays were correlated for all the drugs tested except atovaquone. The two enzymatic assays are non-radioactive, rapid, reliable, inexpensive to perform and semi-automatic. However, they do require an initial parasitaemia of 2% with a haematocrit of 1.8%.

Plasmodium falciparum merozoite protein-1 genetic diversity and multiplicity of infection in isolates from Congolese children consulting in a pediatric hospital in Brazzaville.

PubMed

Gueye, Nerly Shirère Gampio; Ntoumi, Francine; Vouvoungui, Christevy; Kobawila, Simon Charles; NKombo, Michael; Mouanga, Alain M; Deibert, Julia; Koukouikila-Koussounda, Felix

2018-07-01

As in many sub-Saharan African countries, the burden of malaria has been reduced in the Republic of Congo as a result of massive deployment of insecticide treated nets and availability of artemisinin-combinations therapies (ACTs). High to moderate genetic diversity of msp-1 gene of Plasmodium falciparum (P. falciparum) has been reported from different parts of the world but limited data are available from Central Africa including the Republic of Congo. For this reason, the aim of study was to investigate the P. falciparum genetic diversity and to determine the multiplicity of infection in P. falciparum isolates from Congolese children in order to dispose of an additional parameter to measure the impact malaria control intervention. A total of 229 blood samples were collected from September 2014 to February 2015 in children aged from one to ten years presenting a paediatric hospital Marien NGOUABI located in Northern part of Brazzaville. Inclusion criterion was fever (axillary temperature ≥ 37.5 °C) or history of fever in the preceding 48 h before inclusion in this study. Then thick and thin blood smears were done to detect malaria parasites, to determine parasite density and to identify plasmodial species. Sub-microscopic infection was detected by PCR using the P. falciparum msp-1 gene as molecular marker. The prevalence of microscopic and sub-microscopic infection in this cohort was 10% and 27.5%, respectively. The K1 allelic family was predominant (45% of isolates) whereas the RO33 and MAD20 represented 35% and 20%, respectively of isolates. In this study 48% (38/79) of isolates harbored more than one parasite clone. Overall the multiplicity of infection (MOI) was 1.7. According to type of infection, the MOI was significantly higher in children with microscopic infection (2.5 vs 1.4 for submicroscopic infection, P = .001). When considering age, hemoglobin genotype (AA or AS) and level and parasite density, no association was observed with the MOI

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Hsin-Ying; Chang, An-Chi; Wang, Chia-Chi

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. Themore » 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.« less

Mitochondrial population genomic analyses reveal population structure and demography of Indian Plasmodium falciparum.

PubMed

Tyagi, Suchi; Das, Aparup

2015-09-01

Inference on the genetic diversity of Plasmodium falciparum populations could help in better management of malaria. A very recent study with mitochondrial (mt) genomes in global P. falciparum had revealed interesting evolutionary genetic patterns of Indian isolates in comparison to global ones. However, no population genetic study using the whole mt genome sequences of P. falciparum isolates collected in the entire distribution range in India has yet been performed. We herewith have analyzed 85 whole mt genomes (48 already published and 37 entirely new) sampled from eight differentially endemic Indian locations to estimate genetic diversity and infer population structure and historical demography of Indian P. falciparum. We found 19 novel Indian-specific Single Nucleotide Polymorphisms (SNPs) and 22 novel haplotypes segregating in Indian P. falciparum. Accordingly, high haplotype and nucleotide diversities were detected in Indian P. falciparum in comparison to many other global isolates. Indian P. falciparum populations were found to be moderately sub-structured with four different genetic clusters. Interestingly, group of local populations aggregate to form each cluster; while samples from Jharkhand and Odisha formed a single cluster, P. falciparum isolates from Asom formed an independent one. Similarly, Surat, Bilaspur and Betul formed a single cluster and Goa and Mangalore formed another. Interestingly, P. falciparum isolates from the two later populations were significantly genetically differentiated from isolates collected in other six Indian locations. Signature of historical population expansion was evident in five population samples, and the onset of expansion event was found to be very similar to African P. falciparum. In agreement with the previous finding, the estimated Time to Most Recent Common Ancestor (TMRCA) and the effective population size were high in Indian P. falciparum. All these genetic features of Indian P. falciparum with high mt genome

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

Genetic variation of pfhrp2 in Plasmodium falciparum isolates from Yemen and the performance of HRP2-based malaria rapid diagnostic test.

PubMed

Atroosh, Wahib M; Al-Mekhlafi, Hesham M; Al-Jasari, Adel; Sady, Hany; Al-Delaimy, Ahmed K; Nasr, Nabil A; Dawaki, Salwa; Abdulsalam, Awatif M; Ithoi, Init; Lau, Yee Ling; Fong, Mun Yik; Surin, Johari

2015-07-22

The genetic variation in the Plasmodium falciparum histidine-rich protein 2 (pfhrp2) gene that may compromise the use of pfhrp2-based rapid diagnostic tests (RDTs) for the diagnosis of malaria was assessed in P. falciparum isolates from Yemen. This study was conducted in Hodeidah and Al-Mahwit governorates, Yemen. A total of 622 individuals with fever were examined for malaria by CareStart malaria HRP2-RDT and Giemsa-stained thin and thick blood films. The Pfhrp2 gene was amplified and sequenced from 180 isolates, and subjected to amino acid repeat types analysis. A total of 188 (30.2%) participants were found positive for P. falciparum by the RDT. Overall, 12 different amino acid repeat types were identified in Yemeni isolates. Six repeat types were detected in all the isolates (100%) namely types 1, 2, 6, 7, 10 and 12 while types 9 and 11 were not detected in any of the isolates. Moreover, the sensitivity and specificity of the used PfHRP2-based RDTs were high (90.5% and 96.1%, respectively). The present study provides data on the genetic variation within the pfhrp2 gene, and its potential impact on the PfHRP2-based RDTs commonly used in Yemen. CareStart Malaria HRP2-based RDT showed high sensitivity and specificity in endemic areas of Yemen.

Ferrocene-chloroquine analogues as antimalarial agents: in vitro activity of ferrochloroquine against 103 Gabonese isolates of Plasmodium falciparum.

PubMed

Pradines, B; Fusai, T; Daries, W; Laloge, V; Rogier, C; Millet, P; Panconi, E; Kombila, M; Parzy, D

2001-08-01

The in vitro activities of ferrochloroquine, chloroquine, quinine, mefloquine, halofantrine, amodiaquine, primaquine, atovaquone and artesunate were evaluated against Plasmodium falciparum isolates from children with uncomplicated malaria from Libreville (Gabon), using an isotopic, micro, drug susceptibility test. The IC(50) values for ferrochloroquine were in the range 0.43-30.9 nM and the geometric mean IC(50) for the 103 isolates was 10.8 nM (95% CI 8.6-13.5 nM), while the geometric means for chloroquine, quinine, mefloquine, amodiaquine and primaquine were 370 nM, 341 nM, 8.3 nM, 18.1 nM and 7.6 microM, respectively. Ferrochloroquine was active against P. falciparum isolates, 95% of which showed in vitro resistance to chloroquine. Weak positive significant correlations were observed between the responses to ferrochloroquine and that to chloroquine, amodiaquine and quinine, but too low to suggest cross-resistance. There was no significant correlation between the response to ferrochloroquine and those to mefloquine, halofantrine, primaquine, atovaquone or artesunate. Ferrochloroquine may be an important alternative drug for the treatment of chloroquine-resistant malaria.

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

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

Pascual, Aurélie; Fall, Bécaye; Wurtz, Nathalie; Fall, Mansour; Camara, Cheikhou; Nakoulima, Aminata; Baret, Eric; Diatta, Bakary; Fall, Khadidiatou Ba; Mbaye, Pape Saliou; Diémé, Yaya; Bercion, Raymond; Bogreau, Hervé; Briolant, Sébastien; Rogier, Christophe; Wade, Boubacar; Pradines, Bruno

2013-06-07

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. 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. 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). 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 significantly associated with QN susceptibility

Pro-inflammatory Cytokine Response and Genetic Diversity in Merozoite Surface Protein 2 of Plasmodium falciparum Isolates from Nigeria.

PubMed

Ajibaye, Olusola; Osuntoki, Akinniyi A; Ebuehi, Albert Ot; Iwalokun, Bamidele A; Balogun, Emmanuel O; Egbuna, Kathleen N

2017-01-01

Polymorphisms in Plasmodium falciparum merozoite surface protein-2 ( msp -2) and associated parasite genetic diversity which varies between malaria-endemic regions remain a limitation in malaria vaccine development. Pro-inflammatory cytokines are important in immunity against malaria, understanding the influence of genetic diversity on cytokine response is important for effective vaccine design. P. falciparum isolates obtained from 300 Nigerians with uncomplicated falciparum malaria at Ijede General Hospital, Ijede (IJE), General Hospital Ajeromi, Ajeromi (AJE) and Saint Kizito Mission Hospital, Lekki, were genotyped by nested polymerase chain reaction of msp -2 block 3 while ELISA was used to determine the pro-inflammatory cytokine response to describe the genetic diversity of P. falciparum . Eighteen alleles were observed for msp -2 loci. Of the 195 isolates, 61 (31.0%) had only FC27-type alleles, 38 (19.7%) had only 3D7-type alleles, and 49.3% had multiple parasite lines with both alleles. Band sizes were 275-625 bp for FC27 and 150-425 bp for 3D7. Four alleles were observed from LEK, 2 (375-425 bp) and 2 (275-325 bp) of FC27-and 3D7-types, respectively; 12 alleles from AJE, 9 (275-625 bp) and 3 (325-425 bp) of FC27-types and 3D7-types, respectively; while IJE had a total of 12 alleles, 9 (275-625 bp) and 3 (325-425 bp) of FC27-types and 3D7-types, respectively. Mean multiplicity of infection (MOI) was 1.54. Heterozygosity ( H E ) ranged from 0.77 to 0.87 and was highest for IJE (0.87). Cytokine response was higher among <5 years and was significantly associated with MOI ( P > 0.05) but with neither parasite density nor infection type. P. falciparum genetic diversity is extensive in Nigeria, protection via pro-inflammatory cytokines have little or no interplay with infection multiplicity.

False-negative malaria rapid diagnostic tests in Rwanda: impact of Plasmodium falciparum isolates lacking hrp2 and declining malaria transmission.

PubMed

Kozycki, Christina T; Umulisa, Noella; Rulisa, Stephen; Mwikarago, Emil I; Musabyimana, Jean Pierre; Habimana, Jean Pierre; Karema, Corine; Krogstad, Donald J

2017-03-20

Rapid diagnostic tests (RDTs) for histidine rich protein 2 (HRP2) are often used to determine whether persons with fever should be treated with anti-malarials. However, Plasmodium falciparum parasites with a deletion of the hrp2 gene yield false-negative RDTs and there are concerns the sensitivity of HRP2-based RDTs may fall when the intensity of transmission decreases. This observational study enrolled 9226 patients at three health centres in Rwanda from April 2014 to April 2015. It then compared the sensitivity of RDTs based on HRP2 and the Plasmodium lactate dehydrogenase (pLDH) to microscopy (thick smears) for the diagnosis of malaria. PCR was used to determine whether deletions of the histidine-rich central repeat region of the hrp2 gene (exon 2) were associated with false-negative HRP2-based RDTs. In comparison to microscopy, the sensitivity and specificity of HRP2- and pLDH-based RDTs were 89.5 and 86.2% and 80.2 and 94.3%, respectively. When the results for both RDTs were combined, sensitivity rose to 91.8% and specificity was 85.7%. Additionally, when smear positivity fell from 46 to 3%, the sensitivity of the HRP2-based RDT fell from 88 to 67%. Of 370 samples with false-negative HRP2 RDT results for which PCR was performed, 140 (38%) were identified as P. falciparum by PCR. Of the isolates identified as P. falciparum by PCR, 32 (23%) were negative for the hrp2 gene based on PCR. Of the 32 P. falciparum isolates negative for hrp2 by PCR, 17 (53%) were positive based on the pLDH RDT. This prospective study of RDT performance coincided with a decline in the intensity of malaria transmission in Kibirizi (fall in slide positivity from 46 to 3%). This decline was associated with a decrease in HRP2 RDT sensitivity (from 88 to 67%). While P. falciparum isolates without the hrp2 gene were an important cause of false-negative HRP2-based RDTs, most were identified by the pLDH-based RDT. Although WHO does not recommend the use of combined HRP2/pLDH testing in sub

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. © The American Society of Tropical Medicine and Hygiene.

Population genetic structure and natural selection of Plasmodium falciparum apical membrane antigen-1 in Myanmar isolates.

PubMed

Kang, Jung-Mi; Lee, Jinyoung; Moe, Mya; Jun, Hojong; Lê, Hương Giang; Kim, Tae Im; Thái, Thị Lam; Sohn, Woon-Mok; Myint, Moe Kyaw; Lin, Khin; Shin, Ho-Joon; Kim, Tong-Soo; Na, Byoung-Kuk

2018-02-07

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is one of leading blood stage malaria vaccine candidates. However, genetic variation and antigenic diversity identified in global PfAMA-1 are major hurdles in the development of an effective vaccine based on this antigen. In this study, genetic structure and the effect of natural selection of PfAMA-1 among Myanmar P. falciparum isolates were analysed. Blood samples were collected from 58 Myanmar patients with falciparum malaria. Full-length PfAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. PfAMA-1 sequence of each isolate was sequenced. Polymorphic characteristics and effect of natural selection were analysed with using DNASTAR, MEGA4, and DnaSP programs. Polymorphic nature and natural selection in 459 global PfAMA-1 were also analysed. Thirty-seven different haplotypes of PfAMA-1 were identified in 58 Myanmar P. falciparum isolates. Most amino acid changes identified in Myanmar PfAMA-1 were found in domains I and III. Overall patterns of amino acid changes in Myanmar PfAMA-1 were similar to those in global PfAMA-1. However, frequencies of amino acid changes differed by country. Novel amino acid changes in Myanmar PfAMA-1 were also identified. Evidences for natural selection and recombination event were observed in global PfAMA-1. Among 51 commonly identified amino acid changes in global PfAMA-1 sequences, 43 were found in predicted RBC-binding sites, B-cell epitopes, or IUR regions. Myanmar PfAMA-1 showed similar patterns of nucleotide diversity and amino acid polymorphisms compared to those of global PfAMA-1. Balancing natural selection and intragenic recombination across PfAMA-1 are likely to play major roles in generating genetic diversity in global PfAMA-1. Most common amino acid changes in global PfAMA-1 were located in predicted B-cell epitopes where high levels of nucleotide diversity and balancing natural selection were found. These results highlight the

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

PubMed

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

2013-02-01

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. 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. 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. 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). Genetic diversity in P. falciparum field isolates in central Sudan was high and consisted of multiple clones.

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

PFMDR1 POLYMORPHISMS INFLUENCE ON IN VITRO SENSITIVITY OF THAI PLASMODIUM FALCIPARUM ISOLATES TO PRIMAQUINE, SITAMAQUINE AND TAFENOQUINE.

PubMed

Kaewpruk, Napaporn; Tan-ariya, Peerapan; Ward, Stephen A; Sitthichot, Naruemon; Suwandittakul, Nantana; Mungthin, Mathirut

2016-05-01

Primaquine (PQ), an 8-aminoquinoline, is considered a tissue schizonticide drug for radical cure in vivax and ovale malaria, with minimal impact on asexual erythrocytic stages at therapeutic concentrations. Tafenoquine (TQ), a new 8-aminoquinoline analog of PQ, is active against both malaria parasite tissue and blood stages and is being promoted as a drug candidate for antimalarial chemotherapy and chemoprophylaxis and potential transmission blocking against Plasmodium vivax and P. falciparum. This study compared in vitro sensitivity of Thai P. falciparum isolates against three 8-aminoquinolines, PQ, TQ and sitamaquine (SQ), a related 8-aminoquinoline and assessed the importance of pfmdr1 polymorphism on the in vitro response. Seventy-eight laboratory adapted Thai P. falciparum isolates were evaluated for in vitro sensitivity to the three 8-aminoquinolines using a radioisotopic assay, and pfmdr1 polymorphisms were determined using PCR-based methods. All three drugs have weak antiplasmodial activity against asexual erythrocytic stage with SQ being the most potent by almost 10 folds. Cross susceptibility was observed in all three 8-aminoquinolines. Parasites containing pfmdr1 86Y, 184Y or 1034S allele exhibit significantly higher PQ IC₅₀. TQ sensitivity was reduced in those parasites containing pfmdr1 86Y, 1034S or 1042N allele. However, there was no significant influence of pfmdr1 alleles on SQ sensitivity. The data highlight unique differences among three representative 8-aminoquinoline drugs that may be useful in understanding their potential utility in antimalarial development.

Isolation, molecular identification and quinolone-susceptibility testing of Arcobacter spp. isolated from fresh vegetables in Spain.

PubMed

González, Ana; Bayas Morejón, Isidro Favián; Ferrús, María Antonia

2017-08-01

Some species of the Arcobacter genus are considered emerging foodborne and waterborne enteropathogens. However, the presence of Arcobacter spp. in vegetables very little is known, because most studies have focused on foods of animal origin. On the other hand, quinolones are considered as first-line drugs for the treatment of infection by campylobacteria in human patients, but few data are currently available about the resistance levels to these antibiotics among Arcobacter species. Therefore, the aim of this study was to investigate the presence and diversity of arcobacters isolated from fresh vegetables such as lettuces, spinaches, chards and cabbages. Resistance to quinolones of the isolates was also investigated. One hundred fresh vegetables samples purchased from seven local retail markets in Valencia (Spain) during eight months were analysed. The study included 41 lettuces, 21 spinaches, 34 chards and 4 cabbages. Samples were analysed by culture and by molecular methods before and after enrichment. By culture, 17 out of 100 analysed samples were Arcobacter positive and twenty-five isolates were obtained from them. Direct detection by PCR was low, with only 4% Arcobacter spp. positive samples. This percentage increased considerably, up 20%, after 48 h enrichment. By polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), 17 out of the 25 isolates were identified as A. butzleri and 8 as A. cryaerophilus. Only two A. butzleri isolates showed resistance to levofloxacin and ciprofloxacin. The sequencing of a fragment of the QRDR region of the gyrA gene from the quinolones-resistant isolates revealed the presence of a mutation in position 254 of this gene (C-T transition). This study is the first report about the presence of pathogenic species of Arcobacter spp. in chards and cabbages and confirms that fresh vegetables can act as transmission vehicle to humans. Moreover, the presence of A. butzleri quinolone resistant in vegetables could

The Plasmodium falciparum chloroquine resistance transporter is associated with the ex vivo P. falciparum African parasite response to pyronaridine.

PubMed

Madamet, Marylin; Briolant, Sébastien; Amalvict, Rémy; Benoit, Nicolas; Bouchiba, Housem; Cren, Julien; Pradines, Bruno

2016-02-09

The pyronaridine-artesunate combination is one of the most recent oral artemisinin-based therapeutic combinations (ACTs) recommended for the treatment of uncomplicated P. falciparum malaria. The emergence of P. falciparum resistance to artemisinin has recently developed in Southeast Asia. Little data are available on the association between pyronaridine susceptibility and polymorphisms in genes involved in antimalarial drug resistance. The objective of the present study was to investigate the association between ex vivo responses to pyronaridine and the K76T mutation in the pfcrt gene in P. falciparum isolates. The assessment of ex vivo susceptibility to pyronaridine was performed on 296 P. falciparum isolates using a standard 42-h 3H-hypoxanthine uptake inhibition method. The K76T mutation was also investigated. The pyronaridine IC50 (inhibitory concentration 50 %) ranged from 0.55 to 80.0 nM. Ex vivo responses to pyronaridine were significantly associated with the K76T mutation (p-value = 0.020). The reduced susceptibility to pyronaridine, defined as IC50 > 60 nM, was significantly associated with the K76T mutation (p-value = 0.004). Using a Bayesian mixture modelling approach, the pyronaridine IC50 were classified into three components: component A (IC50 median 15.9 nM), component B (IC50 median 34.2 nM) and component C (IC50 median 63.3 nM). The K76T mutation was represented in 46.3% of the isolates in component A, 47.2% of the isolates in component B and 73.3% of the isolates in component C (p-value = 0.021). These results showed the ex vivo reduced susceptibility to pyronaridine, i.e., IC50 > 60 nM, associated with the K76T mutation.

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

[Presence of the double pfmdr1 mutation 86Tyr and 1246 Tyr in clones of a chloroquine-resistant west African isolate of Plasmodium falciparum].

PubMed

Pinheiro, L; Franco, S; Adagu, I S; Rosa, R; Rosário, V E; Warhurst, D C

2003-01-01

Isolates of Plasmodium falciparum from three areas of West Africa were recovered from cryopreservation and their chloroquine-sensitivity were determined in vitro. Of the 90 samples studied, 60 were from Guinea-Bissau (30Resistant/30Sensitive), 15 were from S. Tomé and Príncipe (11Resistant/4Sensitive) and 15 were from Angola (11Resistant/4Sensitive). All the isolates were sensitive to mefloquine. Using the polymerase chain reaction/restriction fragment length polymorphism technique (PCR/RFLP) it was possible to detect two mutations in the pfmdr1 gene, often associated with chloroquine-resistance. 66% of the samples from Guiné-Bissau showed a correlation with chloroquine-resistance while 73% of the samples from São Tomé and Angola altogether had the 86Tyr mutation. The present study on West African isolates and clones showed, for the first time, the presence of a double point mutation in the pfmdr1 gene one being found, up to now, only in South America isolates of Plasmodium falciparum.

Evolution of genetic polymorphisms of Plasmodium falciparum merozoite surface protein (PfMSP) in Thailand.

PubMed

Kuesap, Jiraporn; Chaijaroenkul, Wanna; Ketprathum, Kanchanok; Tattiyapong, Puntanat; Na-Bangchang, Kesara

2014-02-01

Plasmodium falciparum malaria is a major public health problem in Thailand due to the emergence of multidrug resistance. The understanding of genetic diversity of malaria parasites is essential for developing effective drugs and vaccines. The genetic diversity of the merozoite surface protein-1 (PfMSP-1) and merozoite surface protein-2 (PfMSP-2) genes was investigated in a total of 145 P. falciparum isolates collected from Mae Sot District, Tak Province, Thailand during 3 different periods (1997-1999, 2005-2007, and 2009-2010). Analysis of genetic polymorphisms was performed to track the evolution of genetic change of P. falciparum using PCR. Both individual genes and their combination patterns showed marked genetic diversity during the 3 study periods. The results strongly support that P. falciparum isolates in Thailand are markedly diverse and patterns changed with time. These 2 polymorphic genes could be used as molecular markers to detect multiple clone infections and differentiate recrudescence from reinfection in P. falciparum isolates in Thailand.

Characterization of Vancomycin-Resistant Enterococcus faecalis and Enterococcus faecium Isolated from Fresh Produces and Human Fecal Samples.

PubMed

Kim, Min-Chan; Cha, Min-Hyeok; Ryu, Jae-Gee; Woo, Gun-Jo

2017-04-01

Increased enterococcal infections in hospitals and multidrug-resistant and vancomycin-resistant enterococci (VRE) isolated from humans, animals, and food sources raised public health concern on the presence of VRE in multiple sources. We performed a comparative analysis of the antimicrobial resistance and genetics of VRE isolates derived from fresh produce and human fecal samples. Of 389 Enterococcus isolates, 8 fecal and 3 produce isolates were resistant to vancomycin and teicoplanin; all harbored vanA gene. The VRE isolates showed multidrug-resistant properties. The isolates from fresh produce in this study showed to have the common shared characteristics with the isolates from humans by the results of antimicrobial resistance, multilocus sequence typing, and Tn 1546 transposon analysis. Therefore, VRE isolates from fresh produce are likely related to VRE derived from humans. The results suggested that VRE may contaminate vegetables through the environment, and the contaminated vegetables could then act as a vehicle for human infections. Ongoing nationwide surveillance of antibiotic resistance and the promotion of the proper use of antibiotics are necessary.

Ex vivo activity of Proveblue, a methylene blue, against field isolates of Plasmodium falciparum in Dakar, Senegal from 2013-2015.

PubMed

Fall, Bécaye; Madamet, Marylin; Diawara, Silman; Briolant, Sébastien; Wade, Khalifa Ababacar; Lo, Gora; Nakoulima, Aminata; Fall, Mansour; Bercion, Raymond; Kounta, Mame Bou; Amalvict, Rémi; Benoit, Nicolas; Gueye, Mamadou Wague; Diatta, Bakary; Wade, Boubacar; Pradines, Bruno

2017-08-01

Resistance to most antimalarial drugs has spread from Southeast Asia to Africa. Accordingly, new therapies to use with artemisinin-based combination therapy (triple ACT) are urgently needed. Proveblue, a methylene blue preparation, was found to exhibit antimalarial activity against Plasmodium falciparum strains in vitro. Proveblue has synergistic effects when used in combination with dihydroartemisinin, and has been shown to significantly reduce or prevent cerebral malaria in mice. The objectives of the current study were to evaluate the in vitro baseline susceptibility of clinical field isolates to Proveblue, compare its activity with that of other standard antimalarial drugs and define the patterns of cross-susceptibility between Proveblue and conventional antimalarial drugs. The Proveblue IC 50 of 76 P. falciparum isolates ranged from 0.5 nM to 135.1 nM, with a mean of 8.1 nM [95% confidence interval, 6.4-10.3]. Proveblue was found to be more active against P. falciparum parasites than chloroquine, quinine, monodesethylamodiaquine, mefloquine, piperaquine, doxycycline (P <0.001) and lumefantrine (P = 0.014). Proveblue was as active as pyronaridine (P = 0.927), but was less active than dihydroartemisinin and artesunate (P <0.001). The only significant cross-susceptibilities found were between Proveblue and dihydroartemisinin (r 2  = 0.195, P = 0.0001), artesunate (r 2  = 0.187, P = 0.0002) and piperaquine (r 2  = 0.063, P = 0.029). The present study clearly demonstrates the potential of Proveblue as an effective therapeutic agent against P. falciparum. In this context, the use of Proveblue as part of the triple ACT treatment for multidrug-resistant malaria warrants further investigation. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Multiple Phenotypic and Genotypic Artemisinin Sensitivity Evaluation of Malian Plasmodium falciparum Isolates.

PubMed

Niaré, Karamoko; Paloque, Lucie; Ménard, Sandie; Tor, Pety; Ramadani, Arba P; Augereau, Jean-Michel; Dara, Antoine; Berry, Antoine; Benoit-Vical, Françoise; Doumbo, Ogobara K

2018-04-01

We assessed the ex vivo/in vitro sensitivity of 54 Malian Plasmodium falciparum isolates to artemisinin for the monitoring of drug resistance in this area. The artemisinin sensitivity of parasites was evaluated using 1) the ex vivo and in vitro parasite recrudescence detection after treatment of the ring stage with 1-200 nM artemisinin for 48 hours and 2) the in vitro parasite recrudescence kinetics assay over 7 days after 6-hour treatment of the ring stage with 700 nM dihydroartemisinin (DHA). In addition, as recommended by the World Health Organization for artemisinin resistance characterization, the ring-stage survival assay (RSA 0-3 h ) was performed and the parasite isolates were sequenced at the kelch 13 propeller locus. No clinical and molecular evidence of artemisinin resistance was observed. However, these isolates present different phenotypic profiles in response to artemisinin treatments. Despite all RSA 0-3 h values less than 1.5%, six out of 46 (13.0%) isolates tested ex vivo and four out of six (66.7%) isolates tested in vitro were able to multiply after 48-hour treatments with 100 nM artemisinin. Moreover, five out of eight isolates tested showed faster parasite recovery after DHA treatment in kinetic assays. The presence of such phenotypes needs to be taken into account in the assessment of the efficacy of artemisinins in Mali. The assays presented here appear as valuable tools for the monitoring of artemisinin sensitivity in the field and thus could help to evaluate the risk of emergence of artemisinin resistance in Africa.

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

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

Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum.

PubMed

Oyebola, Kolapo M; Idowu, Emmanuel T; Olukosi, Yetunde A; Awolola, Taiwo S; Amambua-Ngwa, Alfred

2017-06-29

The burden of falciparum malaria is especially high in sub-Saharan Africa. Differences in pressure from host immunity and antimalarial drugs lead to adaptive changes responsible for high level of genetic variations within and between the parasite populations. Population-specific genetic studies to survey for genes under positive or balancing selection resulting from drug pressure or host immunity will allow for refinement of interventions. We performed a pooled sequencing (pool-seq) of the genomes of 100 Plasmodium falciparum isolates from Nigeria. We explored allele-frequency based neutrality test (Tajima's D) and integrated haplotype score (iHS) to identify genes under selection. Fourteen shared iHS regions that had at least 2 SNPs with a score > 2.5 were identified. These regions code for genes that were likely to have been under strong directional selection. Two of these genes were the chloroquine resistance transporter (CRT) on chromosome 7 and the multidrug resistance 1 (MDR1) on chromosome 5. There was a weak signature of selection in the dihydrofolate reductase (DHFR) gene on chromosome 4 and MDR5 genes on chromosome 13, with only 2 and 3 SNPs respectively identified within the iHS window. We observed strong selection pressure attributable to continued chloroquine and sulfadoxine-pyrimethamine use despite their official proscription for the treatment of uncomplicated malaria. There was also a major selective sweep on chromosome 6 which had 32 SNPs within the shared iHS region. Tajima's D of circumsporozoite protein (CSP), erythrocyte-binding antigen (EBA-175), merozoite surface proteins - MSP3 and MSP7, merozoite surface protein duffy binding-like (MSPDBL2) and serine repeat antigen (SERA-5) were 1.38, 1.29, 0.73, 0.84 and 0.21, respectively. We have demonstrated the use of pool-seq to understand genomic patterns of selection and variability in P. falciparum from Nigeria, which bears the highest burden of infections. This investigation identified known

Antimicrobial resistance in Escherichia coli and Salmonella spp. isolates from fresh produce and the impact to food safety.

PubMed

Vital, Pierangeli G; Caballes, Marie Bernadine D; Rivera, Windell L

2017-09-02

Foodborne diseases associated with fresh produce consumption have escalated worldwide, causing microbial safety of produce of critical importance. Bacteria that have increasingly been detected in fresh produce are Escherichia coli and Salmonella spp., both of which have been shown to progressively display antimicrobial resistance. The study focused on the assessment of antimicrobial resistance of these enteric bacteria from different kinds of fresh produce from various open air markets and supermarkets in the Philippines. Using the disk diffusion assay on a total of 50 bacterial isolates obtained from 410 fresh produce surveyed, monoresistance to tetracycline was observed to be the most prevalent (38%), followed by multidrug resistance to tetracycline, chloramphenicol, ciprofloxacin, and nalidixic acid (4%), and lastly by dual resistance to tetracycline and chloramphenicol (2%). Using multiplex and simplex polymerase chain reaction (PCR) assays, tetA (75%) and tetB (9%) were found in tetracycline resistant isolates, whereas catI (67%) and catIII (33%) were detected in chloramphenicol resistant isolates. Sequence analysis of gyr and par genes from the ciprofloxacin and nalidixic acid resistant isolates revealed different mutations. Based on the results, fresh produce act as a reservoir of these antibiotic resistant bacteria which may pose health threat to consumers.

Direct Comparison of the Histidine-rich Protein-2 Enzyme-linked Immunosorbent Assay (HRP-2 ELISA) and Malaria SYBR Green I Fluorescence (MSF) Drug Sensitivity Tests in Plasmodium falciparum Reference Clones and Fresh ex vivo Field Isolates from Cambodia

DTIC Science & Technology

2013-07-12

assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 1979, 16:710–718. 3. Noedl H, Attlmayr B...40:685–691. 32. Hawley SR, Bray PG, Mungthin M, Atkinson JD, O’Neill PM, Ward SA: Relationship between antimalarial drug activity , accumulation, and...success rate when testing DHA, AS, MQ, QN, CQ, and PPQ activities . A “successful” IC50 assay result for each P. falciparum clinical isolate was defined as

A simple and efficient method for cryopreservation and recovery of viable Plasmodium vivax and P. falciparum sporozoites.

PubMed

Singh, Naresh; Barnes, Samantha J; Jenwithisuk, Rachaneeporn; Sattabongkot, Jetsumon; Adams, John H

2016-10-01

Plasmodium falciparum and Plasmodium vivax sporozoites are the crucial stages of malaria parasites that initiate infection in humans. However, studies to develop new vaccines and drugs targeting these infective stages remain insufficient due to limited availability of sporozoites for research. This is a consequence of relatively few facilities that are established to produce sporozoites of human malaria parasites, sporozoites remaining viable for only a few days, and infected mosquitoes being a biohazard, making them difficult to transport. Cryopreservation of sporozoites offers the potential to alleviate these limitations and enhance sporozoite availability. These experiments were performed to evaluate methods for cryopreservation of P. vivax and P. falciparum sporozoites. Sporozoites, isolated in sterile buffer from infected mosquitoes by manual dissection of salivary glands, were cryopreserved using several types of commercially available serum-free cryoprotective solutions. The efficiency of cryopreservation was validated by a standard in vitro gliding motility assay as a measure of sporozoite activity. Viability of infective sporozoites was defined as percent gliding of sporozoites attached to the coverslip. Significant differences were observed among the cryopreservation media and protocols evaluated, with CryoStor CS2 giving the best results for both P. falciparum and P. vivax, whereas Hestar 200 worked efficiently only for P. vivax sporozoites. Further improvement in recovery of viable sporozoites would be anticipated using automated controlled-rate freezing equipment. Our results demonstrate that cryopreservation provides an alternative for experimental studies that currently rely on fresh P. falciparum and P. vivax sporozoites. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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. Copyright © 2015, American Association for the Advancement of Science.

Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia.

PubMed

Rachid Viana, Giselle Maria; Akinyi Okoth, Sheila; Silva-Flannery, Luciana; Lima Barbosa, Danielle Regina; Macedo de Oliveira, Alexandre; Goldman, Ira F; Morton, Lindsay C; Huber, Curtis; Anez, Arletta; Dantas Machado, Ricardo Luiz; Aranha Camargo, Luís Marcelo; Costa Negreiros do Valle, Suiane; Marins Póvoa, Marinete; Udhayakumar, Venkatachalam; Barnwell, John W

2017-01-01

More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region.

Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia

PubMed Central

Rachid Viana, Giselle Maria; Akinyi Okoth, Sheila; Silva-Flannery, Luciana; Lima Barbosa, Danielle Regina; Macedo de Oliveira, Alexandre; Goldman, Ira F.; Morton, Lindsay C.; Huber, Curtis; Anez, Arletta; Dantas Machado, Ricardo Luiz; Aranha Camargo, Luís Marcelo; Costa Negreiros do Valle, Suiane; Marins Póvoa, Marinete; Barnwell, John W.

2017-01-01

More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region. PMID:28301474

Cellular mechanisms of action and resistance of Plasmodium falciparum to artemisinin.

PubMed

Phompradit, Papichaya; Chaijaroenkul, Wanna; Na-Bangchang, Kesara

2017-12-01

The recent reports of high failure rates and decline in in vitro sensitivity of Plasmodium falciparum to artemisinin-based combination therapies (ACTs) suggest the possibility of clinical artemisinin resistance along the Thai-Cambodian and Thai-Myanmar borders. The study investigated cellular mechanisms of action and resistance of P. falciparum to artesunate (stage specific activity, interaction with hemozoin, and anti-oxidant levels) in the two paired P. falciparum isolates (MSF046 and MSF060) collected before treatment with a 3-day artesunate-mefloquine and at the time of recrudescence. In addition, the link of these cellular mechanisms to the polymorphisms of the candidate artemisinin-resistant genes (pfatp6, pfcrt, pfmdr1, pfmrp1, and K13 propeller) was also investigated. Morphological change was observed in both pairs of the primary and recrudesced P. falciparum isolates during 12-48 h of exposure to artesunate (at IC 90 ). A marked decrease in parasite viability was found in the recrudesced isolates of both MSF046 and MSD060. The extent of the reduction (% change of baseline) in total glutathione concentrations was significantly lower in recrudesced (32.1 and 1.7%) compared with primary (45.5 and 53.7%) isolates of both MSF046 and MSF060. The extent of reduction of hemozoin content in MSF046 was significantly higher in the recrudesced (76.8%) isolate compared with the primary isolate (99.5%). For MSF060 on the other hand, increase in hemozoin content was found in the recrudesced isolate and the extent of such increase was significantly higher in recrudesced (93.1%) than the primary isolate (87.5%). Polymorphism of K13 (N458Y) together with pfmdr1 copy number correlated well with sensitivity of both isolates to artesunate. Results of this preliminary study suggests possible role of glutathione-dependent detoxification system as well as heme degradation as cellular mechanisms of action and resistance of artemisinins.

Genetic characterization of an epidemic of Plasmodium falciparum malaria among Yanomami Amerindians.

PubMed

Laserson, K F; Petralanda, I; Almera, R; Barker, R H; Spielman, A; Maguire, J H; Wirth, D F

1999-12-01

Malaria parasites are genetically diverse at all levels of endemicity. In contrast, the merozoite surface protein (MSP) alleles in samples from 2 isolated populations of Yanomami Amerindians during an epidemic of Plasmodium falciparum were identical. The nonvariable restriction fragment length polymorphism patterns further suggested that the sequential outbreak comprised only a single P. falciparum genotype. By examination of serial samples from single human infections, the MSP characteristics were found to remain constant throughout the course of infection. An apparent clonal population structure of parasites seemed to cause outbreaks in small isolated villages. The use of standard molecular epidemiologic methods to measure genetic diversity in malaria revealed the occurrence of a genetically monomorphic population of P. falciparum within a human community.

FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand

PubMed Central

Tome-Garcia, Jessica; Doetsch, Fiona; Tsankova, Nadejda M.

2018-01-01

Direct isolation of human neural and glioma stem cells from fresh tissues permits their biological study without prior culture and may capture novel aspects of their molecular phenotype in their native state. Recently, we demonstrated the ability to prospectively isolate stem cell populations from fresh human germinal matrix and glioblastoma samples, exploiting the ability of cells to bind the Epidermal Growth Factor (EGF) ligand in fluorescence-activated cell sorting (FACS). We demonstrated that FACS-isolated EGF-bound neural and glioblastoma populations encompass the sphere-forming colonies in vitro, and are capable of both self-renewal and multilineage differentiation. Here we describe in detail the purification methodology of EGF-bound (i.e., EGFR+) human neural and glioma cells with stem cell properties from fresh postmortem and surgical tissues. The ability to prospectively isolate stem cell populations using native ligand-binding ability opens new doors for understanding both normal and tumor cell biology in uncultured conditions, and is applicable for various downstream molecular sequencing studies at both population and single-cell resolution. PMID:29516026

Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Honduras.

PubMed

Lopez, Ana Cecilia; Ortiz, Andres; Coello, Jorge; Sosa-Ochoa, Wilfredo; Torres, Rosa E Mejia; Banegas, Engels I; Jovel, Irina; Fontecha, Gustavo A

2012-11-26

Understanding the population structure of Plasmodium species through genetic diversity studies can assist in the design of more effective malaria control strategies, particularly in vaccine development. Central America is an area where malaria is a public health problem, but little is known about the genetic diversity of the parasite's circulating species. This study aimed to investigate the allelic frequency and molecular diversity of five surface antigens in field isolates from Honduras. Five molecular markers were analysed to determine the genotypes of Plasmodium vivax and Plasmodium falciparum from endemic areas in Honduras. Genetic diversity of ama-1, msp-1 and csp was investigated for P. vivax, and msp-1 and msp-2 for P. falciparum. Allelic frequencies were calculated and sequence analysis performed. A high genetic diversity was observed within Plasmodium isolates from Honduras. A different number of genotypes were elucidated: 41 (n = 77) for pvama-1; 23 (n = 84) for pvcsp; and 23 (n = 35) for pfmsp-1. Pvcsp sequences showed VK210 as the only subtype present in Honduran isolates. Pvmsp-1 (F2) was the most polymorphic marker for P. vivax isolates while pvama-1 was least variable. All three allelic families described for pfmsp-1 (n = 30) block 2 (K1, MAD20, and RO33), and both allelic families described for the central domain of pfmsp-2 (n = 11) (3D7 and FC27) were detected. However, K1 and 3D7 allelic families were predominant. All markers were randomly distributed across the country and no geographic correlation was found. To date, this is the most complete report on molecular characterization of P. vivax and P. falciparum field isolates in Honduras with regards to genetic diversity. These results indicate that P. vivax and P. falciparum parasite populations are highly diverse in Honduras despite the low level of transmission.

Plasmodium falciparum and Plasmodium vivax Demonstrate Contrasting Chloroquine Resistance Reversal Phenotypes.

PubMed

Wirjanata, Grennady; Handayuni, Irene; Prayoga, Pak; Leonardo, Leo; Apriyanti, Dwi; Trianty, Leily; Wandosa, Ruland; Gobay, Basbak; Kenangalem, Enny; Poespoprodjo, Jeanne Rini; Noviyanti, Rintis; Kyle, Dennis E; Cheng, Qin; Price, Ric N; Marfurt, Jutta

2017-08-01

High-grade chloroquine (CQ) resistance has emerged in both Plasmodium falciparum and P. vivax The aim of the present study was to investigate the phenotypic differences of CQ resistance in both of these species and the ability of known CQ resistance reversal agents (CQRRAs) to alter CQ susceptibility. Between April 2015 and April 2016, the potential of verapamil (VP), mibefradil (MF), L703,606 (L7), and primaquine (PQ) to reverse CQ resistance was assessed in 46 P. falciparum and 34 P. vivax clinical isolates in Papua, Indonesia, where CQ resistance is present in both species, using a modified schizont maturation assay. In P. falciparum , CQ 50% inhibitory concentrations (IC 50 s) were reduced when CQ was combined with VP (1.4-fold), MF (1.2-fold), L7 (4.2-fold), or PQ (1.8-fold). The degree of CQ resistance reversal in P. falciparum was highly correlated with CQ susceptibility for all CQRRAs ( R 2 = 0.951, 0.852, 0.962, and 0.901 for VP, MF, L7, and PQ, respectively), in line with observations in P. falciparum laboratory strains. In contrast, no reduction in the CQ IC 50 s was observed with any of the CQRRAs in P. vivax , even in those isolates with high chloroquine IC 50 s. The differential effect of CQRRAs in P. falciparum and P. vivax suggests significant differences in CQ kinetics and, potentially, the likely mechanism of CQ resistance between these two species. © Crown copyright 2017.

Assessment of copy number variation in genes related to drug resistance in Plasmodium vivax and Plasmodium falciparum isolates from the Brazilian Amazon and a systematic review of the literature.

PubMed

Costa, Gabriel Luíz; Amaral, Lara Cotta; Fontes, Cor Jesus Fernandes; Carvalho, Luzia Helena; de Brito, Cristiana Ferreira Alves; de Sousa, Taís Nóbrega

2017-04-19

Parasite resistance to anti-malarials represents a great obstacle for malaria elimination. The majority of studies have investigated the association between single-nucleotide polymorphisms (SNPs) and drug resistance; however, it is becoming clear that the copy number variation (CNV) is also associated with this parasite phenotype. To provide a baseline for molecular surveillance of anti-malarial drug resistance in the Brazilian Amazon, the present study characterized the genetic profile of both markers in the most common genes associated with drug resistance in Plasmodium falciparum and Plasmodium vivax isolates. Additionally, these data were compared to data published elsewhere applying a systematic review of the literature published over a 20-year time period. The genomic DNA of 67 patients infected by P. falciparum and P. vivax from three Brazilian States was obtained between 2002 and 2012. CNV in P. falciparum multidrug resistance gene-1 (pfmdr1), GTP cyclohydrolase 1 (pfgch1) and P. vivax multidrug resistance gene-1 (pvmdr1) were assessed by real-time PCR assays. SNPs in the pfmdr1 and pfcrt genes were assessed by PCR-RFLP. A literature search for studies that analysed CNP in the same genes of P. falciparum and P. vivax was conducted between May 2014 and March 2017 across four databases. All analysed samples of P. falciparum carried only one copy of pfmdr1 or pfgch1. Although the pfcrt K76T polymorphism, a determinant of CQ resistance, was present in all samples genotyped, the pfmdr1 N86Y was absent. For P. vivax isolates, an amplification rate of 20% was found for the pvmdr1 gene. The results of the study are in agreement with the low amplification rates for pfmdr1 gene evidenced in the Americas and Africa, while higher rates have been described in Southeast Asia. For P. vivax, very low rates of amplification for pvmdr1 have been described worldwide, with exceptions in French Guiana, Cambodia, Thailand and Brazil. The present study was the first to evaluate

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

2015-10-20

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.

Cloning of Plasmodium falciparum by single-cell sorting

PubMed Central

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-01-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

Cloning of Plasmodium falciparum by single-cell sorting.

PubMed

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-10-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

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

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

Molecular Surveillance for Multidrug-Resistant Plasmodium falciparum, Cambodia

PubMed Central

Shah, Naman K.; Alker, Alisa P.; Sem, Rithy; Susanti, Agustina Ika; Muth, Sinuon; Maguire, Jason D.; Duong, Socheat; Ariey, Frederic; Meshnick, Steven R.

2008-01-01

We conducted surveillance for multidrug-resistant Plasmodium falciparum in Cambodia during 2004–2006 by assessing molecular changes in pfmdr1. The high prevalence of isolates with multiple pfmdr1 copies found in western Cambodia near the Thai border, where artesunate–mefloquine therapy failures occur, contrasts with isolates from eastern Cambodia, where this combination therapy remains highly effective. PMID:18826834

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

Molecular epidemiology of malaria in Cameroon. XV. Experimental studies on serum substitutes and supplements and alternative culture media for in vitro drug sensitivity assays using fresh isolates of Plasmodium falciparum.

PubMed

Basco, Leonardo K

2003-08-01

In vitro drug sensitivity assay is an important tool for various on-going studies aiming to establish the correlation between candidate molecular markers for drug resistance and drug response in laboratory-adapted strains and field isolates of Plasmodium falciparum. A widespread use of this technique in the field would require a suitable substitute that can replace human serum. In this study, several alternative sources of serum substitutes and supplements were evaluated for their capacity to sustain parasite growth for a single life cycle and their compatibility with in vitro assays for clinical isolates that have not been adapted to in vitro culture. Albumax, a commercial preparation of lipid-enriched bovine albumin, did not support parasite growth as much as human serum and fetal calf serum in several isolates. Other serum supplements (AmnioMax and Ultroser) supported parasite growth relatively well. The 50% inhibitory concentrations (IC50s) of chloroquine and antifolates determined with 0.05% Albumax were generally two or three times higher than with human serum. With 10% fetal calf serum, IC50s for chloroquine and antifolates were approximately two times higher and three times lower than with human serum, respectively. The use of AmnioMax and OptiMAb resulted in a greater than two-fold increase in IC50s and several uninterpretable assays. Despite possible batch-to-batch differences, fetal calf serum may be a suitable substitute for in vitro drug assays while awaiting the results of further studies on other serum substitutes and supplements.

Intra coronary freshly isolated bone marrow cells transplantation improve cardiac function in patients with ischemic heart disease

PubMed Central

2012-01-01

Background Autologous bone marrow cell transplantation (BMCs-Tx) is a promising novel option for treatment of cardiovascular disease. In this study we analyzed whether intracoronary autologous freshly isolated BMCs-Tx have beneficial effects on cardiac function in patients with ischemic heart disease (IHD). Results In this prospective nonrandomized study we treated 12 patients with IHD by freshly isolated BMCs-Tx by use of point of care system and compared them with a representative 12 control group without cell therapy. Global ejection fraction (EF) and infarct size area were determined by left ventriculography. Intracoronary transplantation of autologous freshly isolated BMCs led to a significant reduction of infarct size (p < 0.001) and an increase of global EF (p = 0.003) as well as infarct wall movement velocity (p < 0.001) after 6 months follow-up compared to control group. In control group there were no significant differences of global EF, infarct size and infarct wall movement velocity between baseline and 6 months after coronary angiography. Furthermore, we found significant decrease in New York Heart Association (NYHA) as well as significant decrease of B-type natriuretic peptide (BNP) level 6 months after intracoronary cell therapy (p < 0.001), whereas there were no significant differences in control group 6 months after coronary angiography. Conclusions These results demonstrate that intracoronary transplantation of autologous freshly isolated BMCs by use of point of care system is safe and may lead to improvement of cardiac function in patients with IHD. Trial registration Registration number: ISRCTN54510226 PMID:22534049

Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Honduras

PubMed Central

2012-01-01

Background Understanding the population structure of Plasmodium species through genetic diversity studies can assist in the design of more effective malaria control strategies, particularly in vaccine development. Central America is an area where malaria is a public health problem, but little is known about the genetic diversity of the parasite’s circulating species. This study aimed to investigate the allelic frequency and molecular diversity of five surface antigens in field isolates from Honduras. Methods Five molecular markers were analysed to determine the genotypes of Plasmodium vivax and Plasmodium falciparum from endemic areas in Honduras. Genetic diversity of ama-1, msp-1 and csp was investigated for P. vivax, and msp-1 and msp-2 for P. falciparum. Allelic frequencies were calculated and sequence analysis performed. Results and conclusion A high genetic diversity was observed within Plasmodium isolates from Honduras. A different number of genotypes were elucidated: 41 (n = 77) for pvama-1; 23 (n = 84) for pvcsp; and 23 (n = 35) for pfmsp-1. Pvcsp sequences showed VK210 as the only subtype present in Honduran isolates. Pvmsp-1 (F2) was the most polymorphic marker for P. vivax isolates while pvama-1 was least variable. All three allelic families described for pfmsp-1 (n = 30) block 2 (K1, MAD20, and RO33), and both allelic families described for the central domain of pfmsp-2 (n = 11) (3D7 and FC27) were detected. However, K1 and 3D7 allelic families were predominant. All markers were randomly distributed across the country and no geographic correlation was found. To date, this is the most complete report on molecular characterization of P. vivax and P. falciparum field isolates in Honduras with regards to genetic diversity. These results indicate that P. vivax and P. falciparum parasite populations are highly diverse in Honduras despite the low level of transmission. PMID:23181845

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. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum infected erythrocytes

PubMed Central

Saiwaew, Somporn; Sritabal, Juntima; Piaraksa, Nattaporn; Keayarsa, Srisuda; Ruengweerayut, Ronnatrai; Utaisin, Chirapong; Sila, Patima; Niramis, Rangsan; Udomsangpetch, Rachanee; Charunwatthana, Prakaykaew; Pongponratn, Emsri; Pukrittayakamee, Sasithon; Leitgeb, Anna M.; Wahlgren, Mats; Lee, Sue J.; Day, Nicholas P. J.; White, Nicholas J.; Dondorp, Arjen M.; Chotivanich, Kesinee

2017-01-01

In severe falciparum malaria cytoadherence of parasitised red blood cells (PRBCs) to vascular endothelium (causing sequestration) and to uninfected red cells (causing rosette formation) contribute to microcirculatory flow obstruction in vital organs. Heparin can reverse the underlying ligand-receptor interactions, but may increase the bleeding risks. As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Sevuparin has been evaluated recently in patients with uncomplicated falciparum malaria, and is currently investigated in a clinical trial for sickle cell disease. The effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum isolates from Thailand were investigated. Trophozoite stages of P. falciparum-infected RBCs (Pf-iRBCs) were cultured from 49 patients with malaria. Pf-iRBCs were treated with sevuparin at 37°C and assessed in rosetting and in cytoadhesion assays with human dermal microvascular endothelial cells (HDMECs) under static and flow conditions. The proportion of Pf-iRBCs forming rosettes ranged from 6.5% to 26.0% (median = 12.2%). Rosetting was dose dependently disrupted by sevuparin (50% disruption by 250 μg/mL). Overall 57% of P. falciparum isolates bound to HDMECs under static conditions; median (interquartile range) Pf-iRBC binding was 8.5 (3.0–38.0) Pf-iRBCs/1000 HDMECs. Sevuparin in concentrations ≥ 100 μg/mL inhibited cytoadherence. Sevuparin disrupts P. falciparum rosette formation in a dose dependent manner and inhibits cytoadherence to endothelial cells. The data support assessment of sevuparin as an adjunctive treatment to the standard therapy in severe falciparum malaria. PMID:28249043

Antiplasmodial activity of two medicinal plants against clinical isolates of Plasmodium falciparum and Plasmodium berghei infected mice.

PubMed

Attemene, Serge David Dago; Beourou, Sylvain; Tuo, Karim; Gnondjui, Albert Alloh; Konate, Abibatou; Toure, Andre Offianan; Kati-Coulibaly, Seraphin; Djaman, Joseph Alico

2018-03-01

Malaria is an infectious and deadly parasitic disease, associated with fever, anaemia and other ailments. Unfortunately the upsurge of plasmodium multidrug resistant constrained researchers to look for new effective drugs. Medicinal plants seem to be an unquenchable source of bioactive principles in the treatment of various diseases. The aim of this study was to assess the antiplasmodial activity of two Ivorian medicinal plants. The in vitro activity was evaluated against clinical isolates and Plasmodium falciparum K1 multidrug resistant strain using the fluorescence based SYBR green I assay. The in vivo bioassay was carried out using the classical 4 day suppressive and curative tests on Plasmodium berghei infected mice. Results showed that the in vitro bioassay of both plant extracts were found to exhibit a promising and moderate antiparasitic effects on clinical isolates (5 µg/mL < IC 50  < 15 µg/mL) and Plasmodium falciparum multidrug resistant K1 strain (15 µg/mL < IC 50  < 50 µg/mL). Furthermore, the in vivo antiplasmodial screening of both extracts showed a significant decrease in parasitemia, which was dose-dependent. Body temperature in mice treated with both extracts at experimental doses increased, compared to the negative control group and was dose-dependent. As for mice body weight a significant decrease ( p  < 0.001) was noticed in the negative control group compared to tested groups of animals. The hydroethanolic stem bark extract of Anthocleista djalonensis A Chev and leaves extract of Ziziphus mauritiana Lam exhibited anti-malarial activities. Therefore, the bioactive compounds of both plant extracts need to be investigated.

Detection of the Plasmodium falciparum Kelch-13 gene P553L mutation in sporozoites isolated from mosquito salivary glands in South-Central Vietnam.

PubMed

Maeno, Yoshimasa; Quang, Nguyen Tuyen; Culleton, Richard; Kawai, Satoru; Masuda, Gaku; Hori, Kaoru; Nakazawa, Shusuke; Marchand, Ron P

2017-06-24

Plasmodium falciparum has developed resistance against artemisinin in Southeast Asia. Mutations in the P. falciparum Kelch-13 (Pfk13) gene are associated with artemisinin resistance in vitro and in vivo. We investigated the prevalence of mutations in PfK13 from sporozoite-stage parasites isolated from the salivary glands of Anopheles dirus mosquitoes. Mosquitoes were caught by human-landing catches at two locations within the Khanh Phu commune, South-Central Vietnam. Identification of Anopheles species was performed based on morphological features and nucleotide sequence analysis. Sporozoite-infected salivary glands were stored on filter paper and at 4-6 °C. A nested-PCR targeting the small subunit ribosomal RNA gene was used for Plasmodium species identification. Pfk13 was amplified by nested PCR, and subjected to nucleotide sequencing. Five of 33 P. falciparum sporozoite samples carried the P553L mutation at the PfK13 locus. This mutation has been recorded previously in Vietnam, but not in Khanh Hoa province, were surveys of K13 polymorphism have not previously been carried out. These results demonstrate the utility of mosquito-stage malaria parasite samples for studies on the molecular epidemiology of drug resistance.

The requirement for freshly isolated human colorectal cancer (CRC) cells in isolating CRC stem cells.

PubMed

Fan, F; Bellister, S; Lu, J; Ye, X; Boulbes, D R; Tozzi, F; Sceusi, E; Kopetz, S; Tian, F; Xia, L; Zhou, Y; Bhattacharya, R; Ellis, L M

2015-02-03

Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs. Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment. None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not. PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.

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

Methylene blue induced morphological deformations in Plasmodium falciparum gametocytes: implications for transmission-blocking.

PubMed

Wadi, Ishan; Pillai, C Radhakrishna; Anvikar, Anupkumar R; Sinha, Abhinav; Nath, Mahendra; Valecha, Neena

2018-01-08

Malaria remains a global health problem despite availability of effective tools. For malaria elimination, drugs targeting sexual stages of Plasmodium falciparum need to be incorporated in treatment regimen along with schizonticidal drugs to interrupt transmission. Primaquine is recommended as a transmission blocking drug for its effect on mature gametocytes but is not extensively utilized because of associated safety concerns among glucose-6-phosphate dehydrogenase (G6PD) deficient patients. In present work, methylene blue, which is proposed as an alternative to primaquine is investigated for its gametocytocidal activity amongst Indian field isolates. An effort has been made to establish Indian field isolates of P. falciparum as in vitro model for gametocytocidal drugs screening. Plasmodium falciparum isolates were adapted to in vitro culture and induced to gametocyte production by hypoxanthine and culture was enriched for gametocyte stages using N-acetyl-glucosamine. Gametocytes were incubated with methylene blue for 48 h and stage specific gametocytocidal activity was evaluated by microscopic examination. Plasmodium falciparum field isolates RKL-9 and JDP-8 were able to reproducibly produce gametocytes in high yield and were used to screen gametocytocidal drugs. Methylene blue was found to target gametocytes in a concentration dependent manner by either completely eliminating gametocytes or rendering them morphologically deformed with mean IC 50 (early stages) as 424.1 nM and mean IC 50 (late stages) as 106.4 nM. These morphologically altered gametocytes appeared highly degenerated having shrinkage, distortions and membrane deformations. Field isolates that produce gametocytes in high yield in vitro can be identified and used to screen gametocytocidal drugs. These isolates should be used for validation of gametocytocidal hits obtained previously by using lab adapted reference strains. Methylene blue was found to target gametocytes produced from Indian field

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

PubMed

Quashie, Neils B; Duah, Nancy O; Abuaku, Benjamin; Quaye, Lydia; Ayanful-Torgby, Ruth; Akwoviah, George A; Kweku, Margaret; Johnson, Jacob D; Lucchi, Naomi W; Udhayakumar, Venkatachalam; Duplessis, Christopher; Kronmann, Karl C; Koram, Kwadwo A

2013-12-17

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. 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. 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, significant elevation of

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

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.

In-vitro interaction of tafenoquine and chloroquine in Plasmodium falciparum from northwestern Thailand.

PubMed

Vollnberg, Anke; Prajakwong, Somsak; Sirichaisinthop, Jeeraphat; Wiedermann, Gerhard; Wernsdorfer, Gunther; Wernsdorfer, Walther H

2003-01-01

The blood schizontocidal, pharmacodynamic interaction between tafenoquine (WR 238605--a 5-phenoxyprimaquine derivative--and chloroquine was investigated, using an in-vitro test for the inhibition of schizont maturation, in 15 fresh isolates of Plasmodium falciparum that originated from northwestern Thailand and neighbouring Myanmar. In this area the parasite is highly resistant to chloroquine. The geometric mean cut-off concentrations of schizont maturation for tafenoquine and chloroquine were 5261 nM and 7638 nM, respectively. With a mixture of tafenoquine and chloroquine, the mean cut-off concentration was 5252 nM, corresponding to 389 nM tafenoquine + 4863 nM chloroquine. Further analysis showed that the interaction between tafenoquine and chloroquine was additive within the range of EC20 and EC77. At concentrations higher than the EC77, interaction was moderately synergistic. While tafenoquine did not reverse the resistance to chloroquine to the degree of clinically relevant sensitivity, there was evidence that the blood schizontocidal efficacy of tafenoquine would be enhanced in the presence of chloroquine.

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; Dajem, Saad M Bin; 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. Copyright © 2016 Elsevier B.V. All rights reserved.

Global sequence diversity of the lactate dehydrogenase gene in Plasmodium falciparum.

PubMed

Simpalipan, Phumin; Pattaradilokrat, Sittiporn; Harnyuttanakorn, Pongchai

2018-01-09

Antigen-detecting rapid diagnostic tests (RDTs) have been recommended by the World Health Organization for use in remote areas to improve malaria case management. Lactate dehydrogenase (LDH) of Plasmodium falciparum is one of the main parasite antigens employed by various commercial RDTs. It has been hypothesized that the poor detection of LDH-based RDTs is attributed in part to the sequence diversity of the gene. To test this, the present study aimed to investigate the genetic diversity of the P. falciparum ldh gene in Thailand and to construct the map of LDH sequence diversity in P. falciparum populations worldwide. The ldh gene was sequenced for 50 P. falciparum isolates in Thailand and compared with hundreds of sequences from P. falciparum populations worldwide. Several indices of molecular variation were calculated, including the proportion of polymorphic sites, the average nucleotide diversity index (π), and the haplotype diversity index (H). Tests of positive selection and neutrality tests were performed to determine signatures of natural selection on the gene. Mean genetic distance within and between species of Plasmodium ldh was analysed to infer evolutionary relationships. Nucleotide sequences of P. falciparum ldh could be classified into 9 alleles, encoding 5 isoforms of LDH. L1a was the most common allelic type and was distributed in P. falciparum populations worldwide. Plasmodium falciparum ldh sequences were highly conserved, with haplotype and nucleotide diversity values of 0.203 and 0.0004, respectively. The extremely low genetic diversity was maintained by purifying selection, likely due to functional constraints. Phylogenetic analysis inferred the close genetic relationship of P. falciparum to malaria parasites of great apes, rather than to other human malaria parasites. This study revealed the global genetic variation of the ldh gene in P. falciparum, providing knowledge for improving detection of LDH-based RDTs and supporting the candidacy of

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

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

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.

The D113N mutation in the RING E3 ubiquitin protein ligase gene is not associated with ex vivo susceptibility to common anti-malarial drugs in African Plasmodium falciparum isolates.

PubMed

Gendrot, Mathieu; Foguim, Francis Tsombeng; Robert, Marie Gladys; Amalvict, Rémy; Mosnier, Joel; Benoit, Nicolas; Madamet, Marylin; Pradines, Bruno

2018-03-12

Plasmodium falciparum resistance to artemisinin-based combination therapy has emerged and spread in Southeast Asia. In areas where artemisinin resistance is emerging, the efficacy of combination is now based on partner drugs. In this context, the identification of novel markers of resistance is essential to monitor the emergence and spread of resistance to these partner drugs. The ubiquitylation pathway could be a possible target for anti-malarial compounds and might be involved in resistance. Polymorphisms in the E3 ubiquitin-protein ligase (PF3D7_0627300) gene could be associated with decreased in vitro susceptibility to anti-malarial drugs. Plasmodium falciparum isolates were collected from patients hospitalized in France with imported malaria from a malaria-endemic country from January 2015 to December 2016 and, more particularly, from African French-speaking countries. In total, 215 isolates were successfully sequenced for the E3 ubiquitin-protein ligase gene and assessed for ex vivo susceptibility to anti-malarial drugs. The D113N mutation in the RING E3 ubiquitin-protein ligase gene was present in 147 out of the 215 samples (68.4%). The IC 50 values for the ten anti-malarial drugs were not significantly different between the wild-type and mutant parasites (p values between 0.225 and 0.933). There was no significant difference in terms of the percentage of parasites with decreased susceptibility between the D113 wild-type and the 133N mutated P. falciparum strains (p values between 0.541 and 1). The present data confirmed the absence of the association between polymorphisms in the RING E3 ubiquitin-protein ligase gene and the ex vivo susceptibility to common anti-malarial drugs in African P. falciparum isolates.

Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

DTIC Science & Technology

2010-06-17

Pennsylvania State University College of Medicine, Hershey , Pennsylvania, United States of America Abstract Plasmodium falciparum is a highly lethal malaria...www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1000968 Zimmerli S, Edwards S, Ernst JD ( 1996 ) Selective receptor blockade...in field isolates. J Immunol 165: 6341–6346. 22. Baruch DI, Gormely JA, Ma C, Howard RJ, Pasloske BL ( 1996 ) Plasmodium falciparum erythrocyte

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

2016-04-07

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. Copyright © 2016 Kant et al.

A Large Proportion of P. falciparum Isolates in the Amazon Region of Peru Lack pfhrp2 and pfhrp3: Implications for Malaria Rapid Diagnostic Tests

PubMed Central

Gamboa, Dionicia; Ho, Mei-Fong; Bendezu, Jorge; Torres, Katherine; Chiodini, Peter L.; Barnwell, John W.; Incardona, Sandra; Perkins, Mark; Bell, David; McCarthy, James; Cheng, Qin

2010-01-01

Background Malaria rapid diagnostic tests (RDTs) offer significant potential to improve the diagnosis of malaria, and are playing an increasing role in malaria case management, control and elimination. Peru, along with other South American countries, is moving to introduce malaria RDTs as components of malaria control programmes supported by the Global Fund for AIDS, TB and malaria. The selection of the most suitable malaria RDTs is critical to the success of the programmes. Methods Eight of nine microscopy positive P. falciparum samples collected in Iquitos, Peru tested negative or weak positive using HRP2-detecting RDTs. These samples were tested for the presence of pfhrp2 and pfhrp3 and their flanking genes by PCR, as well as the presence of HRP proteins by ELISA. To investigate for geographic extent of HRP-deleted parasites and their temporal occurrence a retrospective study was undertaken on 148 microscopy positive P. falciparum samples collected in different areas of the Amazon region of Peru. Findings Eight of the nine isolates lacked the pfhrp2 and/or pfhrp3 genes and one or both flanking genes, and the absence of HRP was confirmed by ELISA. The retrospective study showed that 61 (41%) and 103 (70%) of the 148 samples lacked the pfhrp2 or pfhrp3 genes respectively, with 32 (21.6%) samples lacking both hrp genes. Conclusions This is the first documentation of P. falciparum field isolates lacking pfhrp2 and/or pfhrp3. The high frequency and wide distribution of different parasites lacking pfhrp2 and/or pfhrp3 in widely dispersed areas in the Peruvian Amazon implies that malaria RDTs targeting HRP2 will fail to detect a high proportion of P. falciparum in malaria-endemic areas of Peru and should not be used. RDTs detecting parasite LDH or aldolase and quality microscopy should be use for malaria diagnosis in this region. There is an urgent need for investigation of the abundance and geographic distribution of these parasites in Peru and neighbouring countries

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.

Genetic polymorphism of merozoite surface protein-1 and merozoite surface protein-2 in Plasmodium falciparum isolates from Brazzaville, Republic of Congo

PubMed Central

2011-01-01

Background The characterization of malaria parasite populations circulating in an area is part of site characterization, as a basis for evaluating the impact of malaria interventions on genetic diversity, parasite species, and multiplicity of infection. The present study was aimed at analysing genetic diversity of Plasmodium falciparum merozoite surface proteins 1 and 2 (MSP-1 and MSP-2) and to determine the multiplicity of infection in clinical isolates collected from children living in the Southern district of Brazzaville in the Republic of Congo. Methods A total of 125 isolates from patients with uncomplicated malaria attending Terinkyo and Madibou health centres were collected between January and June 2005 while evaluating the therapeutic efficacy of amodiaquine-artesunate combination. DNA was extracted and msp-1 and msp-2 genes were genotyped using allele-specific nested-PCR. Results Out of 468 distinct fragments detected, 15 msp-1 and 20 msp-2 genotypes were identified. For the msp-1 gene, K1 family was the predominant allelic type carried alone or in association with RO33 and Mad20 types, whereas the 3D7 family was the most prevalent in the msp-2 gene. Overall, the mean multiplicity of infection was 2.2. Out of 125 samples, 104 (83%) harboured more than one parasite genotype. There was no statistical significant difference in the multiplicity of infection by either sex or age of patients. However, a statistically significant correlation was found between parasite densities and the number of genotypes. Conclusion Polymorphism in P. falciparum clinical isolates from Brazzaville was high and mainly of multiple clones. The basis for the positive association between parasite densities and multiplicity of infection is discussed. PMID:21936949

Enterobacter and Klebsiella species isolated from fresh vegetables marketed in Valencia (Spain) and their clinically relevant resistances to chemotherapeutic agents.

PubMed

Falomir, María Pilar; Rico, Hortensia; Gozalbo, Daniel

2013-12-01

Occurrence of antibiotic-resistant pathogenic or commensal enterobacteria in marketed agricultural foodstuffs may contribute to their incorporation into the food chain and constitutes an additional food safety concern. In this work, we have determined the clinically relevant resistances to 11 common chemotherapeutic agents in Enterobacter and Klebsiella isolates from fresh vegetables from various sources (supermarkets and greengrocers' shops in Valencia, Spain). A total of 96 isolates were obtained from 160 vegetables analyzed (50% positive samples): 68 Enterobacter isolates (59 E. cloacae, two E. aerogenes, two E. cancerogenus, one E. gergoviae, and four E. sakazakii, currently Cronobacter spp.), and 28 Klebsiella isolates (19 K. oxytoca and 9 K. pneumoniae). Only seven isolates were susceptible to all agents tested, and no resistances to ceftazidime, ciprofloxacin, gentamicin, and chloramphenicol were detected. Most isolates were resistant to amoxicillin/clavulanic acid (74 [58 Enterobacter and 16 Klebsiella]) or to ampicillin (80 [55/25]). Other resistances were less frequent: nitrofurantoin (13 isolates [12/1]), tetracycline (6 [5/1]), co-trimoxazole (3 [3/0]), cefotaxime (1 [1/0]), and streptomycin (2 [1/1]). Multiresistant isolates to two (56 [41/15]), three (10 E. cloacae isolates), four (one E. cloacae and one K. pneumoniae isolate), and five (two E. cloacae isolates) chemotherapeutic agents were also detected. The presence of potential pathogens points to marketed fresh produce, which often is eaten raw, as a risk factor for consumer health. In addition, these results support the usefulness of these bacterial species as indicators of the spreading of antibiotic resistances into the environment, particularly in the food chain, and suggest their role as carriers of resistance determinants from farms to consumers, which may constitute an additional "silent" food safety concern. Therefore, there is a need to improve the hygienic quality of marketed fresh

Prevalence, genetic diversity and antimicrobial resistance of Listeria monocytogenes isolated from fresh and smoked fish in Poland.

PubMed

Wieczorek, Kinga; Osek, Jacek

2017-06-01

A total of 57 out of 301 (18.9%) fresh and smoked fish samples in Poland were positive for Listeria monocytotgenes. The bacteria were most frequently identified in fresh and smoked salmon (32.0% and 33.8% respectively) as well as in fresh cod (31.8%). Only three samples of smoked salmon were contaminated with the bacteria above 100 CFU/g. Four molecular serogroups were identified and the most prevalent, 1/2a-3a (40 isolates; 70.2%), was present in samples from both marine (33 strains; 71.7%) and freshwater fish (7 isolates; 63.6%). Similar duality of prevalence was observed only for L. monocytogenes of 1/2b-3b-7 serogroup (14 strains; 24.6%), which was identified in 11 (23.9%) marine and 3 (27.3%) freshwater fish. All isolates harboured 10 virulence-associated genes (inlA, inlB, inlC, inlJ, lmo2672, plcA, plcB, hlyA, actA, and mpl) and most of them (56; 98.2%) also possessed the flaA marker. Several strains displayed resistance to oxacillin (33; 57.9%), ceftriaxone (18; 31.6%), or clindamycin (5; 8.8%), and two isolates of serogroup 1/2a-3a showed multiresistance to all three. Genetic subtyping showed the presence of different pulsotypes belonging to six PFGE clusters. The obtained results provide useful information regarding fish contamination with L. monocytogenes which may have implications for public health. Copyright © 2017 Elsevier Ltd. All rights reserved.

Higher microsatellite diversity in Plasmodium vivax than in sympatric Plasmodium falciparum populations in Pursat, Western Cambodia.

PubMed

Orjuela-Sánchez, Pamela; Sá, Juliana M; Brandi, Michelle C C; Rodrigues, Priscila T; Bastos, Melissa S; Amaratunga, Chanaki; Duong, Socheat; Fairhurst, Rick M; Ferreira, Marcelo U

2013-07-01

Previous microsatellite analyses of sympatric populations of Plasmodium vivax and Plasmodium falciparum in Brazil revealed higher diversity in the former species. However, it remains unclear whether regional species-specific differences in prevalence and transmission levels might account for these findings. Here, we examine sympatric populations of P. vivax (n=87) and P. falciparum (n=164) parasites from Pursat province, Western Cambodia, where both species are similarly prevalent. Using 10 genome-wide microsatellites for P. falciparum and 13 for P. vivax, we found that the P. vivax population was more diverse than the sympatric P. falciparum population (average virtual heterozygosity [HE], 0.87 vs. 0.66, P=0.003), with more multiple-clone infections (89.6% vs. 47.6%) and larger mean number of alleles per marker (16.2 vs. 11.1, P=0.07). Both populations showed significant multi-locus linkage disequilibrium suggestive of a predominantly clonal mode of parasite reproduction. The higher microsatellite diversity found in P. vivax isolates, compared to sympatric P. falciparum isolates, does not necessarily result from local differences in transmission level and may reflect differences in population history between species or increased mutation rates in P. vivax. Copyright © 2013 Elsevier Inc. All rights reserved.

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

PubMed Central

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

Accelerated functional recovery after skeletal muscle ischemia-reperfusion injury using freshly isolated bone marrow cells.

PubMed

Corona, Benjamin T; Rathbone, Christopher R

2014-05-01

Relatively little information exists regarding the usefulness of bone marrow-derived cells for skeletal muscle ischemia-reperfusion injury (I/R), especially when compared with I/R that occurs in other tissues. The objectives of this study were to evaluate the ability of freshly isolated bone marrow cells to home to injured skeletal muscle and to determine their effects on muscle regeneration. Freshly isolated lineage-depleted bone marrow cells (Lin(-) BMCs) were injected intravenously 2 d after I/R. Bioluminescent imaging was used to evaluate cell localization for up to 28 d after injury. Muscle function, the percentage of fibers with centrally located nuclei, and the capillary-to-fiber ratio were evaluated 14 d after delivery of either saline (Saline) or saline containing Lin(-) BMCs (Lin(-) BMCs). Bioluminescence was higher in the injured leg than the contralateral control leg for up to 7 d after injection (P < 0.05) suggestive of cell homing to the injured skeletal muscle. Fourteen days after injury, there was a significant improvement in maximal tetanic torque (40% versus 22% deficit; P < 0.05), a faster rate of force production (+dP/dt) (123.6 versus 94.5 Nmm/S; P < 0.05), and a reduction in the percentage of fibers containing centrally located nuclei (40 versus 17%; P < 0.05), but no change in the capillary-to-fiber ratio in the Lin(-) BMC as compared with the Saline group. The homing of freshly isolated BMCs to injured skeletal muscle after I/R is associated with an increase in functional outcomes. Published by Elsevier Inc.

Bronchodilatory effect of deep inspiration in freshly isolated sheep lungs.

PubMed

Wong, William D; Wang, Lu; Paré, Peter D; Seow, Chun Y

2017-02-01

Taking a big breath is known to reverse bronchoconstriction induced by bronchochallenge in healthy subjects; this bronchodilatory effect of deep inspiration (DI) is diminished in asthmatics. The mechanism underlying the DI effect is not clear. Observations from experiments using isolated airway smooth muscle (ASM) preparations and airway segments suggest that straining of ASM due to DI could lead to bronchodilation, possibly due to strain-induced reduction in ASM contractility. However, factors external to the lung cannot be excluded as potential causes for the DI effect. Neural reflex initiated by stretch receptors in the lung are known to inhibit the broncho-motor tone and enhance vasodilatation; the former directly reduces airway resistance, and the latter facilitates removal of contractile agonists through the bronchial circulation. If the DI effect is solely mediated by factors extrinsic to the lung, the DI effect would be absent in isolated, nonperfused lungs. Here we examined the DI effect in freshly isolated, nonperfused sheep lungs. We found that imposition of DI on isolated lungs resulted in significant bronchodilation, that this DI effect was present only after the lungs were challenged with a contractile agonist (acetylcholine or histamine), and that the effect was independent of the difference in lung volume observed pre- and post-DI. We conclude that a significant portion of the bronchodilatory DI effect stems from factors internal to the lung related to the activation of ASM. Copyright © 2017 the American Physiological Society.

Multinormal In Vitro Distribution Model Suitable for the Distribution of Plasmodium falciparum Chemosusceptibility to Doxycycline▿

PubMed Central

Briolant, Sébastien; Baragatti, Meili; Parola, Philippe; Simon, Fabrice; Tall, Adama; Sokhna, Cheikh; Hovette, Philippe; Mamfoumbi, Modeste Mabika; Koeck, Jean-Louis; Delmont, Jean; Spiegel, André; Castello, Jacky; Gardair, Jean Pierre; Trape, Jean Francois; Kombila, Maryvonne; Minodier, Philippe; Fusai, Thierry; Rogier, Christophe; Pradines, Bruno

2009-01-01

The distribution and range of 50% inhibitory concentrations (IC50s) of doxycycline were determined for 747 isolates obtained between 1997 and 2006 from patients living in Senegal, Republic of the Congo, and Gabon and patients hospitalized in France for imported malaria. The statistical analysis was designed to answer the specific question of whether Plasmodium falciparum has different phenotypes of susceptibility to doxycycline. A triple normal distribution was fitted to the data using a Bayesian mixture modeling approach. The IC50 geometric mean ranged from 6.2 μM to 11.1 μM according to the geographical origin, with a mean of 9.3 μM for all 747 parasites. The values for all 747 isolates were classified into three components: component A, with an IC50 mean of 4.9 μM (±2.1 μM [standard deviation]); component B, with an IC50 mean of 7.7 μM (±1.2 μM); and component C, with an IC50 mean of 17.9 μM (±1.4 μM). According to the origin of the P. falciparum isolates, the triple normal distribution was found in each subgroup. However, the proportion of isolates predicted to belong to component B was most important in isolates from Gabon and Congo and in isolates imported from Africa (from 46 to 56%). In Senegal, 55% of the P. falciparum isolates were predicted to be classified as component C. The cutoff of reduced susceptibility to doxycycline in vitro was estimated to be 35 μM. PMID:19047651

Magnetic isolation of Plasmodium falciparum schizonts iRBCs to generate a high parasitaemia and synchronized in vitro culture.

PubMed

Mata-Cantero, Lydia; Lafuente, Maria J; Sanz, Laura; Rodriguez, Manuel S

2014-03-21

The establishment of methods for an in vitro continuous culture of Plasmodium falciparum is essential for gaining knowledge into its biology and for the development of new treatments. Previously, several techniques have been used to synchronize, enrich and concentrate P. falciparum, although obtaining cultures with high parasitaemia continues being a challenging process. Current methods produce high parasitaemia levels of synchronized P. falciparum cultures by frequent changes of culture medium or reducing the haematocrit. However, these methods are time consuming and sometimes lead to the loss of synchrony. A procedure that combines Percoll and sorbitol treatments, the use of magnetic columns, and the optimization of the in vitro culture conditions to reach high parasitaemia levels for synchronized Plasmodium falciparum cultures is described. A new procedure has been established using P. falciparum 3D7, combining previous reported methodologies to achieve in vitro parasite cultures that reach parasitaemia up to 40% at any intra-erythrocytic stage. High parasitaemia levels are obtained only one day after magnetic column purification without compromising the parasite viability and synchrony. The described procedure allows obtaining a large scale synchronized parasite culture at a high parasitaemia with less manipulations than other methods previously described.

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

A simple field kit for the determination of drug susceptibility in Plasmodium falciparum.

PubMed

Nguyen-Dinh, P; Magloire, R; Chin, W

1983-05-01

A field kit has been developed which greatly simplifies the performance of the 48-hour in vitro test for drug resistance in Plasmodium falciparum. The kit uses an easily reconstituted lyophilized culture medium, and requires only a fingerprick blood sample. In parallel tests with 13 isolates of P. falciparum in Haiti, the new technique had a success rate equal to that of the previously described method, with comparable results in terms of parasite susceptibility in vitro to chloroquine and pyrimethamine.

Polymorphisms of the artemisinin resistant marker (K13) in Plasmodium falciparum parasite populations of Grande Comore Island 10 years after artemisinin combination therapy.

PubMed

Huang, Bo; Deng, Changsheng; Yang, Tao; Xue, Linlu; Wang, Qi; Huang, Shiguang; Su, Xin-zhuan; Liu, Yajun; Zheng, Shaoqin; Guan, Yezhi; Xu, Qin; Zhou, Jiuyao; Yuan, Jie; Bacar, Afane; Abdallah, Kamal Said; Attoumane, Rachad; Mliva, Ahamada M S A; Zhong, Yanchun; Lu, Fangli; Song, Jianping

2015-12-15

Plasmodium falciparum malaria is a significant public health problem in Comoros, and artemisinin combination therapy (ACT) remains the first choice for treating acute uncomplicated P. falciparum. The emergence and spread of artemisinin-resistant P. falciparum in Southeast Asia, associated with mutations in K13-propeller gene, poses a potential threat to ACT efficacy. Detection of mutations in the P. falciparum K13-propeller gene may provide the first-hand information on changes in parasite susceptibility to artemisinin. The objective of this study is to determinate the prevalence of mutant K13-propeller gene among the P. falciparum isolates collected from Grande Comore Island, Union of Comoros, where ACT has been in use since 2004. A total of 207 P. falciparum clinical isolates were collected from the island during March 2006 and October 2007 (n = 118) and March 2013 and December 2014 (n = 89). All isolates were analysed for single nucleotide polymorphisms (SNPs) and haplotypes in the K13-propeller gene using nested PCR and DNA sequencing. Only three 2006-2007 samples carried SNPs in the K13-propeller gene, one having a synonymous (G538G) and the other having two non-synonymous (S477Y and D584E) substitutions leading to two mutated haplotypes (2.2%, 2/95). Three synonymous mutations (R471R, Y500Y, and G538G) (5.9%, 5/85) and 7 non-synonymous substitutions (21.2%, 18/85) with nine mutated haplotypes (18.8%, 16/85) were found in isolates from 2013 to 2014. However, none of the polymorphisms associated with artemisinin-resistance in Southeast Asia was detected from any of the parasites examined. This study showed increased K13-propeller gene diversity among P. falciparum populations on the Island over the course of 8 years (2006-2014). Nevertheless, none of the polymorphisms known to be associated with artemisinin resistance in Asia was detected in the parasite populations examined. Our data suggest that P. falciparum populations in Grande Comore are still

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

PubMed

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

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. 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. 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. The absence of mutations in the K13 gene indicates that P. falciparum parasite populations in the study area are still fully susceptible to artemisinins. © A. Ogouyèmi-Hounto et al., published by EDP Sciences, 2016.

Isolation of Shiga toxin-producing Escherichia coli from fresh produce using STEC heart infusion washed blood agar with mitomycin-C.

PubMed

Lin, Andrew; Nguyen, Lam; Clotilde, Laurie M; Kase, Julie A; Son, Insook; Lauzon, Carol R

2012-11-01

The ability to detect and isolate Shiga toxin-producing Escherichia coli (STEC) remains a major challenge for food microbiologists. Although methods based on nucleic acids and antibodies have improved detection of STECs in foods, isolation of these bacteria remains arduous. STEC isolation is necessary for matching food, environmental, and clinical isolates during outbreak investigations and for distinguishing between pathogenic and nonpathogenic organisms. STEC heart infusion washed blood agar with mitomycin-C (SHIBAM) is a modification of washed sheep blood agar prepared by adding mitomycin-C and optimizing both the washed blood and base agar to better isolate STECs. Most STEC isolates produce a zone of hemolysis on SHIBAM plates and are easily distinguishable from background microbiota. Here, we present data supporting the use of SHIBAM to isolate STECs from fresh produce. SHIBAM was tested for accuracy in identifying STECs (365 of 410 STEC strains were hemolytic, and 63 of 73 E. coli strains that did not produce Shiga toxin were not hemolytic) and for recovery from artificially inoculated fresh produce (11 of 24 romaine lettuce samples and 6 of 24 tomato samples). STEC recovery with SHIBAM agar was greatly improved when compared with recovery on Levine's eosin-methylene blue agar as a reference method.

Conserved mosquito/parasite interactions affect development of Plasmodium falciparum in Africa.

PubMed

Mendes, Antonio M; Schlegelmilch, Timm; Cohuet, Anna; Awono-Ambene, Parfait; De Iorio, Maria; Fontenille, Didier; Morlais, Isabelle; Christophides, George K; Kafatos, Fotis C; Vlachou, Dina

2008-05-16

In much of sub-Saharan Africa, the mosquito Anopheles gambiae is the main vector of the major human malaria parasite, Plasmodium falciparum. Convenient laboratory studies have identified mosquito genes that affect positively or negatively the developmental cycle of the model rodent parasite, P. berghei. Here, we use transcription profiling and reverse genetics to explore whether five disparate mosquito gene regulators of P. berghei development are also pertinent to A. gambiae/P. falciparum interactions in semi-natural conditions, using field isolates of this parasite and geographically related mosquitoes. We detected broadly similar albeit not identical transcriptional responses of these genes to the two parasite species. Gene silencing established that two genes affect similarly both parasites: infections are hindered by the intracellular local activator of actin cytoskeleton dynamics, WASP, but promoted by the hemolymph lipid transporter, ApoII/I. Since P. berghei is not a natural parasite of A. gambiae, these data suggest that the effects of these genes have not been drastically altered by constant interaction and co-evolution of A. gambiae and P. falciparum; this conclusion allowed us to investigate further the mode of action of these two genes in the laboratory model system using a suite of genetic tools and infection assays. We showed that both genes act at the level of midgut invasion during the parasite's developmental transition from ookinete to oocyst. ApoII/I also affects the early stages of oocyst development. These are the first mosquito genes whose significant effects on P. falciparum field isolates have been established by direct experimentation. Importantly, they validate for semi-field human malaria transmission the concept of parasite antagonists and agonists.

Decreasing pfmdr1 Copy Number Suggests that Plasmodium falciparum in Western Cambodia Is Regaining In Vitro Susceptibility to Mefloquine

PubMed Central

Lim, Pharath; Dek, Dalin; Try, Vorleak; Sreng, Sokunthea; Suon, Seila

2015-01-01

Dihydroartemisinin-piperaquine is the current frontline artemisinin combination therapy (ACT) for Plasmodium falciparum malaria in Cambodia but is now failing in several western provinces. To investigate artesunate plus mefloquine (AS+MQ) as a replacement ACT, we measured the prevalence of multiple pfmdr1 copies—a molecular marker for MQ resistance—in 844 P. falciparum clinical isolates collected in 2008 to 2013. The pfmdr1 copy number is decreasing in Western Cambodia, suggesting that P. falciparum is regaining in vitro susceptibility to MQ. PMID:25712365

Isolation and molecular characterization of Vibrio parahaemolyticus from fresh, low-temperature preserved, dried, and salted seafood products in two coastal areas of eastern China.

PubMed

Yang, Zhen-Quan; Jiao, Xin-An; Zhou, Xiao-Hui; Cao, Guo-Xiang; Fang, Wei-Ming; Gu, Rui-Xia

2008-07-31

A total of 1293 seafood samples from fishing farm, retail markets, restaurants and cooking rooms of hotels in Jiangsu province and Shanghai city of China were collected and analyzed for the prevalence of Vibrio parahaemolyticus during July to October in 2007. Two hundred and fifty one isolates of V. parahaemolyticus were identified, of which 8 isolates were positive for tdh and 2 were positive for trh gene. Three tdh positive isolates were identified from low-temperature preserved seafood samples and 5 isolates from fresh seafood samples, of these tdh positive isolates, 3 were positive in ORF8-PCR test. The genetic diversity among V. parahaemolyticus isolates was assessed using random amplified polymorphic DNA (RAPD)-PCR and the results showed that there were 33 different genetic patterns that were clustered into nine groups (groups A to I) at 82% similarity level. About 31.9% of the isolates belong to type III9d that were widely distributed in fresh, iced, frozen, dried and salted seafood samples. Seven tdh positive isolates belonged to group A and one belonged to group C, 2 trh positive isolates were type I10d belonging to group F, which was identical to that of reference strains isolated from patients. This study demonstrated genetic variability within V. parahaemolyticus isolates from seafood in Chinese markets and confirmed the presence of toxigenic V. parahaemolyticus not only in fresh but also in iced and frozen seafood products indicating that low-temperature preserved seafood might be also a vehicle for transmitting pathogenic V. parahaemolyticus.

Simultaneous detection of Plasmodium vivax and Plasmodium falciparum gametocytes in clinical isolates by multiplex-nested RT-PCR

PubMed Central

2012-01-01

Background Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. Methods A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. Results The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. Conclusions The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa. PMID:22682065

Simultaneous detection of Plasmodium vivax and Plasmodium falciparum gametocytes in clinical isolates by multiplex-nested RT-PCR.

PubMed

Kuamsab, Napaporn; Putaporntip, Chaturong; Pattanawong, Urassaya; Jongwutiwes, Somchai

2012-06-10

Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa.

An Adjustable Gas-Mixing Device to Increase Feasibility of In Vitro Culture of Plasmodium falciparum Parasites in the Field

PubMed Central

Volkman, Sarah K.; Ahouidi, Ambroise D.; Ndiaye, Daouda; Mboup, Souleymane; Wirth, Dyann F.

2014-01-01

A challenge to conducting high-impact and reproducible studies of the mechanisms of P. falciparum drug resistance, invasion, virulence, and immunity is the lack of robust and sustainable in vitro culture in the field. While the technology exists and is routinely utilized in developed countries, various factors–from cost, to supply, to quality–make it hard to implement in malaria endemic countries. Here, we design and rigorously evaluate an adjustable gas-mixing device for the in vitro culture of P. falciparum parasites in the field to circumvent this challenge. The device accurately replicates the gas concentrations needed to culture laboratory isolates, short-term adapted field isolates, cryopreserved previously non-adapted isolates, as well as to adapt ex vivo isolates to in vitro culture in the field. We also show an advantage over existing alternatives both in cost and in supply. Furthermore, the adjustable nature of the device makes it an ideal tool for many applications in which varied gas concentrations could be critical to culture success. This adjustable gas-mixing device will dramatically improve the feasibility of in vitro culture of Plasmodium falciparum parasites in malaria endemic countries given its numerous advantages. PMID:24603696

Cytoadhesion to gC1qR through Plasmodium falciparum Erythrocyte Membrane Protein 1 in Severe Malaria

PubMed Central

Magallón-Tejada, Ariel; Machevo, Sónia; Cisteró, Pau; Lavstsen, Thomas; Aide, Pedro; Jiménez, Alfons; Turner, Louise; Gupta, Himanshu; De Las Salas, Briegel; Mandomando, Inacio; Wang, Christian W.; Petersen, Jens E. V.; Muñoz, Jose; Gascón, Joaquim; Macete, Eusebio; Alonso, Pedro L.; Chitnis, Chetan E.

2016-01-01

Cytoadhesion of Plasmodium falciparum infected erythrocytes to gC1qR has been associated with severe malaria, but the parasite ligand involved is currently unknown. To assess if binding to gC1qR is mediated through the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, we analyzed by static binding assays and qPCR the cytoadhesion and var gene transcriptional profile of 86 P. falciparum isolates from Mozambican children with severe and uncomplicated malaria, as well as of a P. falciparum 3D7 line selected for binding to gC1qR (Pf3D7gC1qR). Transcript levels of DC8 correlated positively with cytoadhesion to gC1qR (rho = 0.287, P = 0.007), were higher in isolates from children with severe anemia than with uncomplicated malaria, as well as in isolates from Europeans presenting a first episode of malaria (n = 21) than Mozambican adults (n = 25), and were associated with an increased IgG recognition of infected erythrocytes by flow cytometry. Pf3D7gC1qR overexpressed the DC8 type PFD0020c (5.3-fold transcript levels relative to Seryl-tRNA-synthetase gene) compared to the unselected line (0.001-fold). DBLβ12 from PFD0020c bound to gC1qR in ELISA-based binding assays and polyclonal antibodies against this domain were able to inhibit binding to gC1qR of Pf3D7gC1qR and four Mozambican P. falciparum isolates by 50%. Our results show that DC8-type PfEMP1s mediate binding to gC1qR through conserved surface epitopes in DBLβ12 domain which can be inhibited by strain-transcending functional antibodies. This study supports a key role for gC1qR in malaria-associated endovascular pathogenesis and suggests the feasibility of designing interventions against severe malaria targeting this specific interaction. PMID:27835682

Infectivity of Plasmodium falciparum sporozoites determines emerging parasitemia in infected volunteers.

PubMed

McCall, Matthew B B; Wammes, Linda J; Langenberg, Marijke C C; van Gemert, Geert-Jan; Walk, Jona; Hermsen, Cornelus C; Graumans, Wouter; Koelewijn, Rob; Franetich, Jean-François; Chishimba, Sandra; Gerdsen, Max; Lorthiois, Audrey; van de Vegte, Marga; Mazier, Dominique; Bijker, Else M; van Hellemond, Jaap J; van Genderen, Perry J J; Sauerwein, Robert W

2017-06-21

Malaria sporozoites must first undergo intrahepatic development before a pathogenic blood-stage infection is established. The success of infection depends on host and parasite factors. In healthy human volunteers undergoing controlled human malaria infection (CHMI), we directly compared three clinical Plasmodium falciparum isolates for their ability to infect primary human hepatocytes in vitro and to drive the production of blood-stage parasites in vivo. Our data show a correlation between the efficiency of strain-specific sporozoite invasion of human hepatocytes and the dynamics of patent parasitemia in study subjects, highlighting intrinsic differences in infectivity among P. falciparum isolates from distinct geographical locales. The observed heterogeneity in infectivity among strains underscores the value of assessing the protective efficacy of candidate malaria vaccines against heterologous strains in the CHMI model. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Genetic deletion of HRP2 and HRP3 in Indian Plasmodium falciparum population and false negative malaria rapid diagnostic test.

PubMed

Kumar, Navin; Pande, Veena; Bhatt, R M; Shah, Naman K; Mishra, Neelima; Srivastava, Bina; Valecha, Neena; Anvikar, Anupkumar R

2013-01-01

Genetic polymorphisms in diagnostic antigens are important factors responsible for variable performance of rapid diagnostic tests. Additionally, the failure of antigen expression due to gene deletion may also contribute to variable performance. We report Indian Plasmodium falciparum field isolates lacking both Pfhrp2 and Pfhrp3 genes leading to false negative results of rapid diagnostic tests. The study highlights need to determine the prevalence of P. falciparum isolates lacking these genes in larger field populations in India. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Starzengruber, Peter; Fuehrer, Hans-Peter; Swoboda, Paul; Ganesh, Deepa; Haque, Rashidul; Khan, Wasif A; Graninger, Wolfgang; Noedl, Harald

2014-06-10

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. 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. 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. 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.

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

Base isolation: Fresh insight

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 twomore » 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.« less

Population genetics of GYPB and association study between GYPB*S/s polymorphism and susceptibility to P. falciparum infection in the Brazilian Amazon.

PubMed

Tarazona-Santos, Eduardo; Castilho, Lilian; 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

2011-01-24

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. 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. 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. 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 associated with the GPB S+ variant in this

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.

Molecular mutation profile of pfcrt in Plasmodium falciparum isolates imported from Africa in Henan province.

PubMed

Zhou, Rui-Min; Zhang, Hong-Wei; Yang, Cheng-Yun; Liu, Ying; Zhao, Yu-Ling; Li, Su-Hua; Qian, Dan; Xu, Bian-Li

2016-05-10

Anti-malarial drug resistance is a primary public health problem. Haplotypes of pfcrt gene have been implicated to be molecular markers of chloroquine (CQ) resistance. This study aims to explore the prevalence of polymorphisms in pfcrt in Plasmodium falciparum-infected patients imported from Africa in Henan province. Blood samples were collected from 502 patients who were infected with P. falciparum returning from Africa in Henan province during 2012-2015. The single nucleotide polymorphisms in pfcrt (codons 72-76) were assessed by nested PCR with DNA sequencing and restriction digestion, the haplotype prevalences were also determined. Four haplotypes coding 72-76 of pfcrt were found including CVMNK (wild type), CVIET (mutation type), CVIEK (mutation type), and CV M/I N/E/D/K K/T (mixed type), with 61.95 % (311/502), 33.07 % (166/502), 0.20 % (1/502), and 4.78 % (24/502) prevalence, respectively. Except mixed type, CVIET and CVIEK were the largest proportion of the mutant type in West Africa, accounting for 44.83 % (91/203), followed by East Africa (8/21, 38.10 %), North Africa (4/11, 36.36 %), Central Africa (36/135, 26.67 %), and South Africa (28/132, 21.21 %). There was significant difference among the groups (χ(2) = 23.78, P < 0.05). Mixed type was the largest proportion in North Africa (9.09 %), followed by Central Africa (6.67 %), East Africa (4.76 %), South Africa (4.55 %), and West Africa (3.45 %). There was no significant difference among the groups (χ(2) = 2.31, P > 0.05). The position 72 and 73 of pfcrt showed predominance for the wild type with rates of 100 % (502/502). This study identified four haplotypes of pfcrt in P. falciparum-infected patients imported from Africa in Henan province. The prevalence of mutations in the pfcrt was dropped comparing with other people's researches. It establishes fundamental data for detection of P. falciparum CQR with molecular markers for the imported P. falciparum in China, and it also

Plasmodium falciparum msp1 and msp2 genetic diversity and allele frequencies in parasites isolated from symptomatic malaria patients in Bobo-Dioulasso, Burkina Faso.

PubMed

Somé, Anyirékun Fabrice; Bazié, Thomas; Zongo, Issaka; Yerbanga, R Serge; Nikiéma, Frédéric; Neya, Cathérine; Taho, Liz Karen; Ouédraogo, Jean-Bosco

2018-05-30

In Burkina Faso, malaria remains the overall leading cause of morbidity and mortality accounting for 35.12% of consultations, 40.83% of hospitalizations and 37.5% of deaths. Genotyping of malaria parasite populations remains an important tool to determine the types and number of parasite clones in an infection. The present study aimed to evaluate the merozoite surface protein 1 (msp1) and merozoite surface protein 2 (msp2) genetic diversity and allele frequencies in Bobo-Dioulasso, Burkina Faso. Dried blood spots (DBS) were collected at baseline from patients with uncomplicated malaria in urban health centers in Bobo-Dioulasso. Parasite DNA was extracted using chelex-100 and species were identified using nested PCR. Plamodium falciparum msp1 and msp2 genes were amplified by nested polymerase chain reaction (PCR) and PCR products were analyzed by electrophoresis on a 2.5% agarose gel. Alleles were categorized according to their molecular weight. A total of 228 blood samples were analyzed out of which 227 (99.9%) were confirmed as P. falciparum-positive and one sample classified as mixed infection for P. malaria and P. falciparum. In msp1, the K1 allelic family was predominant with 77.4% (162/209) followed respectively by the MAD20 allelic family with 41.3% and R033 allelic family with 36%. In msp2, the 3D7 allelic family was the most frequently detected with 93.1 % compared to FC27 with 41.3%. Twenty-one different alleles were observed in msp1 with 9 alleles for K1, 8 alleles for MAD20 and 4 alleles for R033. In msp2, 25 individual alleles were detected with 10 alleles for FC27 and 15 alleles for 3D7. The mean multiplicity of falciparum infection was 1.95 with respectively 1.8 (1.76-1.83) and 2.1 (2.03-2.16) for msp1 and msp2 (P = 0.01). Our study showed high genetic diversity and allelic frequencies of msp1 and msp2 in Plasmodium falciparum isolates from symptomatic malaria patients in Bobo-Dioulasso.

Size and sequence polymorphisms in the glutamate-rich protein gene of the human malaria parasite Plasmodium falciparum in Thailand.

PubMed

Pattaradilokrat, Sittiporn; Trakoolsoontorn, Chawinya; Simpalipan, Phumin; Warrit, Natapot; Kaewthamasorn, Morakot; Harnyuttanakorn, Pongchai

2018-01-22

The glutamate-rich protein (GLURP) of the malaria parasite Plasmodium falciparum is a key surface antigen that serves as a component of a clinical vaccine. Moreover, the GLURP gene is also employed routinely as a genetic marker for malarial genotyping in epidemiological studies. While extensive size polymorphisms in GLURP are well recorded, the extent of the sequence diversity of this gene is rarely investigated. The present study aimed to explore the genetic diversity of GLURP in natural populations of P. falciparum. The polymorphic C-terminal repetitive R2 region of GLURP sequences from 65 P. falciparum isolates in Thailand were generated and combined with the data from 103 worldwide isolates to generate a GLURP database. The collection was comprised of 168 alleles, encoding 105 unique GLURP subtypes, characterized by 18 types of amino acid repeat units (AAU). Of these, 28 GLURP subtypes, formed by 10 AAU types, were detected in P. falciparum in Thailand. Among them, 19 GLURP subtypes and 2 AAU types are described for the first time in the Thai parasite population. The AAU sequences were highly conserved, which is likely due to negative selection. Standard Fst analysis revealed the shared distributions of GLURP types among the P. falciparum populations, providing evidence of gene flow among the different demographic populations. Sequence diversity causing size variations in GLURP in Thai P. falciparum populations were detected, and caused by non-synonymous substitutions in repeat units and some insertion/deletion of aspartic acid or glutamic acid codons between repeat units. The P. falciparum population structure based on GLURP showed promising implications for the development of GLURP-based vaccines and for monitoring vaccine efficacy.

The best source of isolated stromal cells for the artificial ovary: medulla or cortex, cryopreserved or fresh?

PubMed

Soares, M; Sahrari, K; Chiti, M C; Amorim, C A; Ambroise, J; Donnez, J; Dolmans, M-M

2015-07-01

What is the best source of ovarian cells for the artificial ovary: medulla or cortex, cryopreserved or fresh? Ovarian cells from fresh medullary tissue, which can be isolated in larger numbers, show higher viability and are able to improve graft vascularization. In a previous study, addition of endothelial cells along with ovarian cells was found to be crucial for formation of a well-vascularized ovary-like structure. This study is the first to evaluate both the effect of cryopreservation and the source of ovarian tissue on isolated ovarian cells. Prospective experimental study in an academic research unit using ovarian tissue from seven patients undergoing surgery for benign gynecologic disease. Ovarian tissue was retrieved from seven patients, with one half processed as fresh (fresh group) and the other half frozen and thawed before processing (frozen group). In each group, ovarian cells from the cortex and medulla were isolated separately, and their viability was tested using a calcein AM/ethidium homodimer viability assay. Fifty thousand cells were then encapsulated in fibrin and grafted to peritoneal pockets in nude mice (14 in all). Grafts recovered after 7 days were analyzed by immunohistochemistry for the presence of ovarian cells (vimentin), proliferation (Ki67) and graft vascularization (double CD34). Cell apoptosis was analyzed by TUNEL assay. Cryopreservation decreased ovarian cell yield (-2804 cells/mg, P = 0.015) and viability (-9.72%, P = 0.052) before grafting and had a considerable (5-fold, P = 0.2) but non-significant negative impact on ovarian cell presence in grafts. The medulla yielded many more cells (+3841 cells/mg, P < 0.001) with higher viability (+18.23%, P < 0.001) than did the cortex. Moreover, grafts with cells from the medulla exhibited a statistically significant 6.44- and 2.47-fold increase in human and total vascular surface area, respectively. P-values were adjusted for multiple testing using the Benjamini-Hochberg method to

Genetic evidence for contribution of human dispersal to the genetic diversity of EBA-175 in Plasmodium falciparum.

PubMed

Yasukochi, Yoshiki; Naka, Izumi; Patarapotikul, Jintana; Hananantachai, Hathairad; Ohashi, Jun

2015-08-01

The 175-kDa erythrocyte binding antigen (EBA-175) of Plasmodium falciparum plays a crucial role in merozoite invasion into human erythrocytes. EBA-175 is believed to have been under diversifying selection; however, there have been no studies investigating the effect of dispersal of humans out of Africa on the genetic variation of EBA-175 in P. falciparum. The PCR-direct sequencing was performed for a part of the eba-175 gene (regions II and III) using DNA samples obtained from Thai patients infected with P. falciparum. The divergence times for the P. falciparum eba-175 alleles were estimated assuming that P. falciparum/Plasmodium reichenowi divergence occurred 6 million years ago (MYA). To examine the possibility of diversifying selection, nonsynonymous and synonymous substitution rates for Plasmodium species were also estimated. A total of 32 eba-175 alleles were identified from 131 Thai P. falciparum isolates. Their estimated divergence time was 0.13-0.14 MYA, before the exodus of humans from Africa. A phylogenetic tree for a large sequence dataset of P. falciparum eba-175 alleles from across the world showed the presence of a basal Asian-specific cluster for all P. falciparum sequences. A markedly more nonsynonymous substitutions than synonymous substitutions in region II in P. falciparum was also detected, but not within Plasmodium species parasitizing African apes, suggesting that diversifying selection has acted specifically on P. falciparum eba-175. Plasmodium falciparum eba-175 genetic diversity appeared to increase following the exodus of Asian ancestors from Africa. Diversifying selection may have played an important role in the diversification of eba-175 allelic lineages. The present results suggest that the dispersals of humans out of Africa influenced significantly the molecular evolution of P. falciparum EBA-175.

Genetic Characterisation of Plasmodium falciparum Isolates with Deletion of the pfhrp2 and/or pfhrp3 Genes in Colombia: The Amazon Region, a Challenge for Malaria Diagnosis and Control

PubMed Central

Dorado, Erika Jimena; Okoth, Sheila Akinyi; Montenegro, Lidia Madeline; Diaz, Gustavo; Barnwell, John W.; Udhayakumar, Venkatachalam; Murillo Solano, Claribel

2016-01-01

Most Plasmodium falciparum-detecting rapid diagnostic tests (RDTs) target histidine-rich protein 2 (PfHRP2). However, P. falciparum isolates with deletion of the pfhrp2 gene and its homolog gene, pfhrp3, have been detected. We carried out an extensive investigation on 365 P. falciparum dried blood samples collected from seven P. falciparum endemic sites in Colombia between 2003 and 2012 to genetically characterise and geographically map pfhrp2- and/or pfhrp3-negative P. falciparum parasites in the country. We found a high proportion of pfhrp2-negative parasites only in Amazonas (15/39; 38.5%), and these parasites were also pfhrp3-negative. These parasites were collected between 2008 and 2009 in Amazonas, while pfhrp3-negative parasites (157/365, 43%) were found in all the sites and from each of the sample collection years evaluated (2003 to 2012). We also found that all pfhrp2- and/or pfhrp3-negative parasites were also negative for one or both flanking genes. Six sub-population clusters were established with 93.3% (14/15) of the pfhrp2-negative parasites grouped in the same cluster and sharing the same haplotype. This haplotype corresponded with the genetic lineage BV1, a multidrug resistant strain that caused two outbreaks reported in Peru between 2010 and 2013. We found this BV1 lineage in the Colombian Amazon as early as 2006. Two new clonal lineages were identified in these parasites from Colombia: the genetic lineages EV1 and F. PfHRP2 sequence analysis revealed high genetic diversity at the amino acid level, with 17 unique sequences identified among 53 PfHRP2 sequences analysed. The use of PfHRP2-based RDTs is not recommended in Amazonas because of the high proportion of parasites with pfhrp2 deletion (38.5%), and implementation of new strategies for malaria diagnosis and control in Amazonas must be prioritised. Moreover, studies to monitor and genetically characterise pfhrp2-negative P. falciparum parasites in the Americas are warranted, given the extensive

Isolation of lactic acid bacteria from pao cai, a Chinese traditional fermented vegetable, with inhibitory activity against Salmonella associated with fresh-cut apple, using a modelling study.

PubMed

Luo, W; Chen, M; Chen, A; Dong, W; Hou, X; Pu, B

2015-04-01

To isolate lactic acid bacteria (LAB) from pao cai, a Chinese traditional fermented vegetable, with outstanding inhibitory activity against Salmonella inoculated on fresh-cut apple, using a modelling method. Four kinds of pao cai were selected. A total of 122 isolates exhibited typical LAB characteristics: Gram-positive and catalase negative, among which 104 (85·24%) colonies showed antibacterial activity against Salmonella by the well diffusion assay. Four colonies showing maximum antibacterial radius against Salmonella were selected to co-inoculate with Salmonella on fresh-cut apple and stored at 10°C, further identified as three strains of Lactobacillus plantarum and one strain of Lactobacillus brevis by 16s rRNA gene sequence analysis. The modified Gompertz model was employed to analyse the growth of the micro-organisms on apple wedges. Two of the four selected strains showed antagonistic activity against Salmonella on fresh-cut apple, one of which, RD1, exhibited best inhibitory activity (Salmonella were greatly inhibited when co-inoculated with RD1 at 10°C at 168 h). No deterioration in odour or appearance of the apple piece was observed by the triangle test when fresh-cut apple was inoculated with RD1. The mathematical modelling method is essential to select LAB with outstanding inhibitory activity against Salmonella associated with fresh-cut apple. LAB RD1 holds promise for the preservation of fresh-cut apple. This study provided a new method on fresh-cut product preservation. Besides, to make the LAB isolating procedure a more correct one, this study first added the mathematical modelling method to the isolating procedure. © 2014 The Society for Applied Microbiology.

Investigating the activity of quinine analogues versus chloroquine resistant Plasmodium falciparum.

PubMed

Dinio, Theresa; Gorka, Alexander P; McGinniss, Andrew; Roepe, Paul D; Morgan, Jeremy B

2012-05-15

Plasmodium falciparum, the deadliest malarial parasite species, has developed resistance against nearly all man-made antimalarial drugs within the past century. However, quinine (QN), the first antimalarial drug, remains efficacious worldwide. Some chloroquine resistant (CQR) P. falciparum strains or isolates show mild cross resistance to QN, but many do not. Further optimization of QN may provide a well-tolerated therapy with improved activity versus CQR malaria. Thus, using the Heck reaction, we have pursued a structure-activity relationship study, including vinyl group modifications of QN. Certain derivatives show good antiplasmodial activity in QN-resistant and QN-sensitive strains, with lower IC(50) values relative to QN. Copyright © 2012 Elsevier Ltd. All rights reserved.

Life-span of in vitro differentiated Plasmodium falciparum gametocytes.

PubMed

Gebru, Tamirat; Lalremruata, Albert; Kremsner, Peter G; Mordmüller, Benjamin; Held, Jana

2017-08-11

The sexual stages (gametocytes) of Plasmodium falciparum do not directly contribute to the pathology of malaria but are essential for transmission of the parasite from the human host to the mosquito. Mature gametocytes circulate in infected human blood for several days and their circulation time has been modelled mathematically from data of previous in vivo studies. This is the first time that longevity of gametocytes is studied experimentally in vitro. The in vitro longevity of P. falciparum gametocytes of 1 clinical isolate and 2 laboratory strains was assessed by three different methods: microscopy, flow cytometry and reverse transcription quantitative real-time PCR (RT-qPCR). Additionally, the rate of gametocytogenesis of the used P. falciparum strains was compared. The maximum in vitro lifespan of P. falciparum gametocytes reached almost 2 months (49 days by flow cytometry, 46 days by microscopy, and at least 52 days by RT-qPCR) from the starting day of gametocyte culture to death of last parasite in the tested strains with an average 50% survival rate of 6.5, 2.6 and 3.5 days, respectively. Peak gametocytaemia was observed on average 19 days after initiation of gametocyte culture followed by a steady decline due to natural decay of the parasites. The rate of gametocytogenesis was highest in the NF54 strain. Plasmodium falciparum mature gametocytes can survive up to 16-32 days (at least 14 days for mature male gametocytes) in vitro in absence of the influence of host factors. This confirms experimentally a previous modelling estimate that used molecular tools for gametocyte detection in treated patients. The survival time might reflect the time the parasite can be transmitted to the mosquito after clearance of asexual parasites. These results underline the importance of efficient transmission blocking agents in the fight against malaria.

In vitro adaptation of Plasmodium falciparum reveal variations in cultivability.

PubMed

White, John; Mascarenhas, Anjali; Pereira, Ligia; Dash, Rashmi; Walke, Jayashri T; Gawas, Pooja; Sharma, Ambika; Manoharan, Suresh Kumar; Guler, Jennifer L; Maki, Jennifer N; Kumar, Ashwani; Mahanta, Jagadish; Valecha, Neena; Dubhashi, Nagesh; Vaz, Marina; Gomes, Edwin; Chery, Laura; Rathod, Pradipsinh K

2016-01-22

Culture-adapted Plasmodium falciparum parasites can offer deeper understanding of geographic variations in drug resistance, pathogenesis and immune evasion. To help ground population-based calculations and inferences from culture-adapted parasites, the complete range of parasites from a study area must be well represented in any collection. To this end, standardized adaptation methods and determinants of successful in vitro adaption were sought. Venous blood was collected from 33 P. falciparum-infected individuals at Goa Medical College and Hospital (Bambolim, Goa, India). Culture variables such as whole blood versus washed blood, heat-inactivated plasma versus Albumax, and different starting haematocrit levels were tested on fresh blood samples from patients. In vitro adaptation was considered successful when two four-fold or greater increases in parasitaemia were observed within, at most, 33 days of attempted culture. Subsequently, parasites from the same patients, which were originally cryopreserved following blood draw, were retested for adaptability for 45 days using identical host red blood cells (RBCs) and culture media. At a new endemic area research site, ~65% of tested patient samples, with varied patient history and clinical presentation, were successfully culture-adapted immediately after blood collection. Cultures set up at 1% haematocrit and 0.5% Albumax adapted most rapidly, but no single test condition was uniformly fatal to culture adaptation. Success was not limited by low patient parasitaemia nor by patient age. Some parasites emerged even after significant delays in sample processing and even after initiation of treatment with anti-malarials. When 'day 0' cryopreserved samples were retested in parallel many months later using identical host RBCs and media, speed to adaptation appeared to be an intrinsic property of the parasites collected from individual patients. Culture adaptation of P. falciparum in a field setting is formally shown to be

Genetic micro-epidemiology of malaria in Papua Indonesia: Extensive P. vivax diversity and a distinct subpopulation of asymptomatic P. falciparum infections.

PubMed

Pava, Zuleima; Noviyanti, Rintis; Handayuni, Irene; Trimarsanto, Hidayat; Trianty, Leily; Burdam, Faustina H; Kenangalem, Enny; Utami, Retno A S; Tirta, Yusrifar K; Coutrier, Farah; Poespoprodjo, Jeanne R; Price, Ric N; Marfurt, Jutta; Auburn, Sarah

2017-01-01

Genetic analyses of Plasmodium have potential to inform on transmission dynamics, but few studies have evaluated this on a local spatial scale. We used microsatellite genotyping to characterise the micro-epidemiology of P. vivax and P. falciparum diversity to inform malaria control strategies in Timika, Papua Indonesia. Genotyping was undertaken on 713 sympatric P. falciparum and P. vivax isolates from a cross-sectional household survey and clinical studies conducted in Timika. Standard population genetic measures were applied, and the data was compared to published data from Kalimantan, Bangka, Sumba and West Timor. Higher diversity (HE = 0.847 vs 0.625; p = 0.017) and polyclonality (46.2% vs 16.5%, p<0.001) were observed in P. vivax versus P. falciparum. Distinct P. falciparum substructure was observed, with two subpopulations, K1 and K2. K1 was comprised solely of asymptomatic infections and displayed greater relatedness to isolates from Sumba than to K2, possibly reflecting imported infections. The results demonstrate the greater refractoriness of P. vivax versus P. falciparum to control measures, and risk of distinct parasite subpopulations persisting in the community undetected by passive surveillance. These findings highlight the need for complimentary new surveillance strategies to identify transmission patterns that cannot be detected with traditional malariometric methods.

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

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

Microsatellite analysis of chloroquine resistance associated alleles and neutral loci reveal genetic structure of Indian Plasmodium falciparum

PubMed Central

Mallick, Prashant K.; Sutton, Patrick L.; Singh, Ruchi; Singh, Om P.; Dash, Aditya P.; Singh, Ashok K.; Carlton, Jane M.; Bhasin, Virendra K.

2013-01-01

Efforts to control malignant malaria caused by Plasmodium falciparum are hampered by the parasite’s acquisition of resistance to antimalarial drugs, e.g., chloroquine. This necessitates evaluating the spread of chloroquine resistance in any malaria-endemic area. India displays highly variable malaria epidemiology and also shares porous international borders with malaria-endemic Southeast Asian countries having multi-drug resistant malaria. Malaria epidemiology in India is believed to be affected by two major factors: high genetic diversity and evolving drug resistance in P. falciparum. How transmission intensity of malaria can influence the genetic structure of chloroquine-resistant P. falciparum population in India is unknown. Here, genetic diversity within and among P. falciparum populations is analyzed with respect to their prevalence and chloroquine resistance observed in 13 different locations in India. Microsatellites developed for P. falciparum, including three putatively neutral and seven microsatellites thought to be under a hitchhiking effect due to chloroquine selection were used. Genetic hitchhiking is observed in five of seven microsatellites flanking the gene responsible for chloroquine resistance. Genetic admixture analysis and F-statistics detected genetically distinct groups in accordance with transmission intensity of different locations and the probable use of chloroquine. A large genetic break between the chloroquine-resistant parasite of the Northeast-East-Island group and Southwest group (FST = 0.253, P<0.001) suggests a long period of isolation or a possibility of different origin between them. A pattern of significant isolation by distance was observed in low transmission areas (r = 0.49, P=0.003, N = 83, Mantel test). An unanticipated pattern of spread of hitchhiking suggests genetic structure for Indian P. falciparum population. Overall, the study suggests that transmission intensity can be an efficient driver for genetic differentiation

Microsatellite analysis of chloroquine resistance associated alleles and neutral loci reveal genetic structure of Indian Plasmodium falciparum.

PubMed

Mallick, Prashant K; Sutton, Patrick L; Singh, Ruchi; Singh, Om P; Dash, Aditya P; Singh, Ashok K; Carlton, Jane M; Bhasin, Virendra K

2013-10-01

Efforts to control malignant malaria caused by Plasmodium falciparum are hampered by the parasite's acquisition of resistance to antimalarial drugs, e.g., chloroquine. This necessitates evaluating the spread of chloroquine resistance in any malaria-endemic area. India displays highly variable malaria epidemiology and also shares porous international borders with malaria-endemic Southeast Asian countries having multi-drug resistant malaria. Malaria epidemiology in India is believed to be affected by two major factors: high genetic diversity and evolving drug resistance in P. falciparum. How transmission intensity of malaria can influence the genetic structure of chloroquine-resistant P. falciparum population in India is unknown. Here, genetic diversity within and among P. falciparum populations is analyzed with respect to their prevalence and chloroquine resistance observed in 13 different locations in India. Microsatellites developed for P. falciparum, including three putatively neutral and seven microsatellites thought to be under a hitchhiking effect due to chloroquine selection were used. Genetic hitchhiking is observed in five of seven microsatellites flanking the gene responsible for chloroquine resistance. Genetic admixture analysis and F-statistics detected genetically distinct groups in accordance with transmission intensity of different locations and the probable use of chloroquine. A large genetic break between the chloroquine-resistant parasite of the Northeast-East-Island group and Southwest group (FST=0.253, P<0.001) suggests a long period of isolation or a possibility of different origin between them. A pattern of significant isolation by distance was observed in low transmission areas (r=0.49, P=0.003, N=83, Mantel test). An unanticipated pattern of spread of hitchhiking suggests genetic structure for Indian P. falciparum population. Overall, the study suggests that transmission intensity can be an efficient driver for genetic differentiation at both

3-Halo Chloroquine Derivatives Overcome Plasmodium falciparum Chloroquine Resistance Transporter-Mediated Drug Resistance in P. falciparum.

PubMed

Edaye, Sonia; Tazoo, Dagobert; Bohle, D Scott; Georges, Elias

2015-12-01

Polymorphism in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) was shown to cause chloroquine resistance. In this report, we examined the antimalarial potential of novel 3-halo chloroquine derivatives (3-chloro, 3-bromo, and 3-iodo) against chloroquine-susceptible and -resistant P. falciparum. All three derivatives inhibited the proliferation of P. falciparum; with 3-iodo chloroquine being most effective. Moreover, 3-iodo chloroquine was highly effective at potentiating and reversing chloroquine toxicity of drug-susceptible and -resistant P. falciparum. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

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

PubMed

Larrañaga, Nerea; Mejía, Rosa E; Hormaza, José I; Montoya, Alberto; Soto, Aida; Fontecha, Gustavo A

2013-10-04

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. 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. There was a low level of genetic diversity in P. falciparum populations from Honduras and Nicaragua. Expected heterozigosity (H(e)) results were similarly low for both populations. A moderate differentiation was revealed by the F(ST) 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.

Endocrine disruption screening by protein and gene expression of vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes.

PubMed

Markell, Lauren K; Mingoia, Robert T; Peterson, Heather M; Yao, Jianhong; Waters, Stephanie M; Finn, James P; Nabb, Diane L; Han, Xing

2014-08-18

Xenobiotics may activate the estrogen receptor, resulting in alteration of normal endocrine functions in animals and humans. Consequently, this necessitates development of assay end points capable of identifying estrogenic xenobiotics. In the present study, we screened the potential estrogenicity of chemicals via their ability to induce vitellogenin (VTG) expression in cultured primary hepatocytes from male trout. A routine method for VTG detection measures the secretion of the protein by enzyme-linked immunosorbent assay (ELISA) in freshly isolated trout hepatocytes. However, this lengthy (6 days) culturing procedure requires that hepatocyte isolation is performed each time the assay is run. We optimized this methodology by investigating the utility of cryopreserved hepatocytes, shortening the incubation time, performing a quantitative real-time PCR (qPCR) method for VTG quantification, and verifying the model system with reference chemicals 17β-estradiol, estrone, diethylstilbestrol, hexestrol, genistein, and a negative control, corticosterone. To test the performance of both freshly isolated and cryopreserved hepatocytes, mRNA was collected from hepatocytes following 24 h treatment for VTG gene expression analysis, whereas cell culture media was collected for a VTG ELISA 96 h post-treatment. EC50 values were obtained for each reference chemical except for corticosterone, which exhibited no induction of VTG gene or protein level. Our results show linear concordance between ELISA and qPCR detection methods. Although there was approximately 50% reduction in VTG inducibility following cryopreservation, linear concordance of EC50 values was found between freshly isolated and cryopreserved hepatocytes, indicating that cryopreservation does not alter the functional assessment of estrogen receptor activation and therefore VTG expression. These studies demonstrate that qPCR is a sensitive and specific method for detecting VTG gene expression that can be used together

Short communication: Characterization of methicillin-resistant Staphylococcus aureus isolated from raw milk fresh cheese in Colombia.

PubMed

Herrera, Fanny C; García-López, María-Luisa; Santos, Jesús A

2016-10-01

The aim of this study was the characterization of a collection of 8 methicillin-resistant Staphylococcus aureus (MRSA) isolates, obtained from samples of fresh cheese (Doble Crema) produced from raw cow milk in small dairies in Colombia. All the isolates harbored the mecA and Panton-Valentine leukocidin (PVL) genes, presented with SCCmec type IV, and belonged to multilocus sequence type 8 and spa type 024. Seven isolates presented 3 closely related pulsed-field gel electrophoresis profiles. Three of them carried the staphylococcal enterotoxin B gene. The isolates were resistant to cefoxitin, oxacillin, penicillin, and ampicillin and susceptible to all non-β-lactams antibiotics tested, with minimum inhibitory concentration values for oxacillin of 4 to 8mg/L. The isolates belonged to the community-acquired MRSA group, suggesting a human source of contamination. The risk of human infection by MRSA via contaminated foods is considered low, but contaminated food commodities can contribute to the worldwide dissemination of clones of community-acquired MRSA. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Photobiomodulation of freshly isolated human adipose tissue-derived stromal vascular fraction cells by pulsed light-emitting diodes for direct clinical application.

PubMed

Priglinger, E; Maier, J; Chaudary, S; Lindner, C; Wurzer, C; Rieger, S; Redl, H; Wolbank, S; Dungel, P

2018-06-01

A highly interesting source for adult stem cells is adipose tissue, from which the stromal vascular fraction (SVF)-a heterogeneous cell population including the adipose-derived stromal/stem cells-can be obtained. To enhance the regenerative potential of freshly isolated SVF cells, low-level light therapy (LLLT) was used. The effects of pulsed blue (475 nm), green (516 nm), and red (635 nm) light from light-emitting diodes applied on freshly isolated SVF were analysed regarding cell phenotype, cell number, viability, adenosine triphosphate content, cytotoxicity, and proliferation but also osteogenic, adipogenic, and proangiogenic differentiation potential. The colony-forming unit fibroblast assay revealed a significantly increased colony size after LLLT with red light compared with untreated cells, whereas the frequency of colony-forming cells was not affected. LLLT with green and red light resulted in a stronger capacity to form vascular tubes by SVF when cultured within 3D fibrin matrices compared with untreated cells, which was corroborated by increased number and length of the single tubes and a significantly higher concentration of vascular endothelial growth factor. Our study showed beneficial effects after LLLT on the vascularization potential and proliferation capacity of SVF cells. Therefore, LLLT using pulsed light-emitting diode light might represent a new approach for activation of freshly isolated SVF cells for direct clinical application. Copyright © 2018 John Wiley & Sons, Ltd.

Platelet-mediated clumping of Plasmodium falciparum infected erythrocytes is associated with high parasitemia but not severe clinical manifestations of malaria in African children

PubMed Central

Arman, Mònica; Raza, Ahmed; Tempest, Louisa J.; Lyke, Kirsten E.; Thera, Mahamadou A.; Koné, Abdoulaye; Plowe, Christopher V.; Doumbo, Ogobara K.; Rowe, J. Alexandra

2009-01-01

Platelet-mediated clumping of Plasmodium falciparum infected erythrocytes is an adhesive phenotype commonly found in field isolates that has previously been associated with severe malaria. Here, clumping was assessed in 131 isolates from Malian children. The clumping phenotype was seen in 6% (n=51) of uncomplicated malaria, 24% (n=51) of severe malaria, and 45% (n=29) of high parasitemia non-severe malaria isolates. Multivariate analysis indicated that clumping was strongly positively associated with parasitemia (F1,122=24.1, p<0.001) but not with disease category (F2,122=1.8, p=0.17). Therefore platelet-mediated clumping in Malian P. falciparum isolates is primarily associated with high parasitemia and not with severe clinical manifestations of malaria. PMID:17984358

Genetic micro-epidemiology of malaria in Papua Indonesia: Extensive P. vivax diversity and a distinct subpopulation of asymptomatic P. falciparum infections

PubMed Central

Pava, Zuleima; Noviyanti, Rintis; Handayuni, Irene; Trimarsanto, Hidayat; Trianty, Leily; Burdam, Faustina H.; Kenangalem, Enny; Utami, Retno A. S.; Tirta, Yusrifar K.; Coutrier, Farah; Poespoprodjo, Jeanne R.; Price, Ric N.; Marfurt, Jutta

2017-01-01

Background Genetic analyses of Plasmodium have potential to inform on transmission dynamics, but few studies have evaluated this on a local spatial scale. We used microsatellite genotyping to characterise the micro-epidemiology of P. vivax and P. falciparum diversity to inform malaria control strategies in Timika, Papua Indonesia. Methods Genotyping was undertaken on 713 sympatric P. falciparum and P. vivax isolates from a cross-sectional household survey and clinical studies conducted in Timika. Standard population genetic measures were applied, and the data was compared to published data from Kalimantan, Bangka, Sumba and West Timor. Results Higher diversity (HE = 0.847 vs 0.625; p = 0.017) and polyclonality (46.2% vs 16.5%, p<0.001) were observed in P. vivax versus P. falciparum. Distinct P. falciparum substructure was observed, with two subpopulations, K1 and K2. K1 was comprised solely of asymptomatic infections and displayed greater relatedness to isolates from Sumba than to K2, possibly reflecting imported infections. Conclusions The results demonstrate the greater refractoriness of P. vivax versus P. falciparum to control measures, and risk of distinct parasite subpopulations persisting in the community undetected by passive surveillance. These findings highlight the need for complimentary new surveillance strategies to identify transmission patterns that cannot be detected with traditional malariometric methods. PMID:28498860

Species Distribution and Prevalence of Putative Virulence Factors in Mesophilic Aeromonas spp. Isolated from Fresh Retail Sushi

PubMed Central

Hoel, Sunniva; Vadstein, Olav; Jakobsen, Anita N.

2017-01-01

Aeromonas spp. are ubiquitous bacteria that have received increasing attention as human pathogens because of their widespread occurrence in food, especially seafood and vegetables. The aim of this work was to assess the species identity and phylogenetic relationship of 118 Aeromonas strains isolated from fresh retail sushi from three producers, and to characterize the isolates with respect to genetic and phenotypic virulence factors. We also evaluate the potential hazard associated with their presence in ready-to-eat seafood not subjected to heat treatment. Mesophilic Aeromonas salmonicida was most prevalent (74%), followed by A. bestiarum (9%), A. dhakensis (5%), A. caviae (5%), A. media (4%), A. hydrophila (2%), and A. piscicola (1%). All isolates were considered potentially pathogenic due to the high prevalence of genes encoding hemolysin (hlyA) (99%), aerolysin (aerA) (98%), cytotoxic enterotoxin (act) (86%), heat-labile cytotonic enterotoxin (alt) (99%), and heat-stable cytotonic enterotoxin (ast) (31%). The shiga-like toxins 1 and 2 (stx-1 and stx-2) were not detected. Moreover, there was heterogeneity in toxin gene distribution among the isolates, and the combination of act/alt/hlyA/aerA was most commonly detected (63%). β-hemolysis was species-dependent and observed in 91% of the isolates. All A. media and A. caviae strains were non-hemolytic. For isolates belonging to this group, lack of hemolysis was possibly related to the absence of the act gene. Swimming motility, linked to adhesion and host invasion, occurred in 65% of the isolates. Partial sequencing of the gyrB gene demonstrated its suitability as a genetic marker for Aeromonas species identification and for assessment of the phylogenetic relationship between the isolates. The gyrB sequence divergence within a given species ranged from 1.3 to 2.9%. A. bestiarum, A. salmonicida, and A. piscicola were the most closely related species; their sequences differed by 2.7–3.4%. The average gyrB sequence

Fibrin hydrogels functionalized with cartilage extracellular matrix and incorporating freshly isolated stromal cells as an injectable for cartilage regeneration.

PubMed

Almeida, H V; Eswaramoorthy, R; Cunniffe, G M; Buckley, C T; O'Brien, F J; Kelly, D J

2016-05-01

Freshly isolated stromal cells can potentially be used as an alternative to in vitro expanded cells in regenerative medicine. Their use requires the development of bioactive hydrogels or scaffolds which provide an environment to enhance their proliferation and tissue-specific differentiation in vivo. The goal of the current study was to develop an injectable fibrin hydrogel functionalized with cartilage ECM microparticles and transforming growth factor (TGF)-β3 as a putative therapeutic for articular cartilage regeneration. ECM microparticles were produced by cryomilling and freeze-drying porcine articular cartilage. Up to 2% (w/v) ECM could be incorporated into fibrin without detrimentally affecting its capacity to form stable hydrogels. To access the chondroinductivity of cartilage ECM, we compared chondrogenesis of infrapatellar fat pad-derived stem cells in fibrin hydrogels functionalized with either particulated ECM or control gelatin microspheres. Cartilage ECM particles could be used to control the delivery of TGF-β3 to IFP-derived stem cells within fibrin hydrogels in vitro, and furthermore, led to higher levels of sulphated glycosaminoglycan (sGAG) and collagen accumulation compared to control constructs loaded with gelatin microspheres. In vivo, freshly isolated stromal cells generated a more cartilage-like tissue within fibrin hydrogels functionalized with cartilage ECM particles compared to the control gelatin loaded constructs. These tissues stained strongly for type II collagen and contained higher levels of sGAGs. These results support the use of fibrin hydrogels functionalized with cartilage ECM components in single-stage, cell-based therapies for joint regeneration. An alternative to the use of in vitro expanded cells in regenerative medicine is the use of freshly isolated stromal cells, where a bioactive scaffold or hydrogel is used to provide an environment that enhances their proliferation and tissue-specific differentiation in vivo. The

ATP release from freshly isolated guinea-pig bladder urothelial cells: a quantification and study of the mechanisms involved.

PubMed

McLatchie, Linda M; Fry, Christopher H

2015-06-01

To quantify the amount of ATP released from freshly isolated bladder urothelial cells, study its control by intracellular and extracellular calcium and identify the pathways responsible for its release. Urothelial cells were isolated from male guinea-pig urinary bladders and stimulated to release ATP by imposition of drag forces by repeated pipetting. ATP was measured using a luciferin-luciferase assay and the effects of modifying internal and external calcium concentration and blockers of potential release pathways studied. Freshly isolated guinea-pig urothelial cells released ATP at a mean (sem) rate of 1.9 (0.1) pmoles/mm(2) cell membrane, corresponding to about 700 pmoles/g of tissue, and about half [49 (6)%, n = 9) of the available cell ATP. This release was reduced to a mean (sem) of 0.46 (0.08) pmoles/mm(2) (160 pmoles/g) with 1.8 mm external calcium, and was increased about two-fold by increasing intracellular calcium. The release from umbrella cells was not significantly different from a mixed intermediate and basal cell population, suggesting that all three groups of cells release a similar amount of ATP per unit area. ATP release was reduced by ≈ 50% by agents that block pannexin and connexin hemichannels. It is suggested that the remainder may involve vesicular release. A significant fraction of cellular ATP is released from isolated urothelial cells by imposing drag forces that cause minimal loss of cell viability. This release involves multiple release pathways, including hemichannels and vesicular release. © 2014 The Authors BJU International © 2014 BJU International.

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

K13 Propeller Alleles, mdr1 Polymorphism, and Drug Effectiveness at Day 3 after Artemether-Lumefantrine Treatment for Plasmodium falciparum Malaria in Colombia, 2014-2015

PubMed Central

Montenegro, Madeline; Neal, Aaron T.; Posada, Maritza; De las Salas, Briegel; Lopera-Mesa, Tatiana M.

2017-01-01

ABSTRACT High treatment failure rates for Plasmodium falciparum malaria have been reported in Colombia for chloroquine, amodiaquine, and sulfadoxine-pyrimethamine. Artemisinin combination therapies were introduced in 2006 in Colombia, where artemether-lumefantrine (AL) is currently used to treat uncomplicated P. falciparum malaria. Artemisinin (ART) resistance was initially observed in Southeast Asia as an increased parasite clearance time, manifesting as a positive thick-blood smear on day 3 after treatment (D3 positivity). Recently, mutations in the propeller domain of the P. falciparum kelch13 gene (K13 propeller) have been associated with ART resistance. In this study, we surveyed AL effectiveness at D3 and molecular markers of drug resistance among 187 uncomplicated P. falciparum cases in 4 regions of Colombia from June 2014 to July 2015. We found that 3.2% (4/125) of patients showed D3 positivity, 100% (163/163) of isolates carried wild-type K13 propeller alleles, 12.9% (23/178) of isolates had multiple copies of the multidrug resistance 1 gene (mdr1), and 75.8% (113/149) of isolates harbored the double mutant NFSDD mdr1 haplotype (the underlining indicates mutant alleles). These data suggest that ART resistance is not currently suspected in Colombia but that monitoring for lumefantrine resistance and AL failures should continue. PMID:28947476

Limited geographical origin and global spread of sulfadoxine-resistant dhps alleles in Plasmodium falciparum populations.

PubMed

Mita, Toshihiro; Venkatesan, Meera; Ohashi, Jun; Culleton, Richard; Takahashi, Nobuyuki; Tsukahara, Takahiro; Ndounga, Mathieu; Dysoley, Lek; Endo, Hiroyoshi; Hombhanje, Francis; Ferreira, Marcelo U; Plowe, Christopher V; Tanabe, Kazuyuki

2011-12-15

Plasmodium falciparum malaria resistant to chloroquine and pyrimethamine originated in limited foci and migrated to Africa. It remains unresolved whether P. falciparum resistance to sulfadoxine, which is conferred by mutations in dihydropteroate synthase (DHPS), evolved following a similar pattern. The dhps locus of 893 P. falciparum isolates from 12 countries in Asia, the Pacific Islands, Africa, and South America was sequenced. Haplotypes of 6 microsatellite loci flanking the dhps locus were determined to define the genetic relationships among sulfadoxine-resistant lineages. Six distinct sulfadoxine-resistant lineages were identified. Highly resistant lineages appear to have originated only in Southeast Asia and South America. Two resistant lineages found throughout Southeast Asia have been introduced to East Africa, where they appear to have spread. The infrequent selection of parasites highly resistant to sulfadoxine and the subsequent migration of resistant lineages from Asia to Africa are similar to the patterns observed in chloroquine and pyrimethamine resistance. These findings strongly suggest that the global migration of resistant parasites has played a decisive role in the establishment of drug-resistant P. falciparum parasites, and that similar patterns may be anticipated for the spread of artemisinin resistance.

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

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.

Molecular analysis demonstrates high prevalence of chloroquine resistance but no evidence of artemisinin resistance in Plasmodium falciparum in the Chittagong Hill Tracts of Bangladesh.

PubMed

Alam, Mohammad Shafiul; Ley, Benedikt; Nima, Maisha Khair; Johora, Fatema Tuj; Hossain, Mohammad Enayet; Thriemer, Kamala; Auburn, Sarah; Marfurt, Jutta; Price, Ric N; Khan, Wasif A

2017-08-15

Artemisinin resistance is present in the Greater Mekong region and poses a significant threat for current anti-malarial treatment guidelines in Bangladesh. The aim of this molecular study was to assess the current status of drug resistance in the Chittagong Hill Tracts of Bangladesh near the Myanmar border. Samples were obtained from patients enrolled into a Clinical Trial (NCT02389374) conducted in Alikadam, Bandarban between August 2014 and January 2015. Plasmodium falciparum infections were confirmed by PCR and all P. falciparum positive isolates genotyped for the pfcrt K76T and pfmdr1 N86Y markers. The propeller region of the kelch 13 (k13) gene was sequenced from isolates from patients with delayed parasite clearance. In total, 130 P. falciparum isolates were available for analysis. The pfcrt mutation K76T, associated with chloroquine resistance was found in 81.5% (106/130) of cases and the pfmdr1 mutation N86Y in 13.9% (18/130) cases. No single nucleotide polymorphisms were observed in the k13 propeller region. This study provides molecular evidence for the ongoing presence of chloroquine resistant P. falciparum in Bangladesh, but no evidence of mutations in the k13 propeller domain associated with artemisinin resistance. Monitoring for artemisinin susceptibility in Bangladesh is needed to ensure early detection and containment emerging anti-malarial resistance.

Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species.

PubMed

Langbeen, A; Jorssen, E P A; Fransen, E; Rodriguez, A P A; García, M Chong; Leroy, J L M R; Bols, P E J

2015-10-01

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 μm for primordial follicles, 47 ± 6.3 μm for primary follicles and 67.1 ± 13.1 μm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

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

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

PubMed

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

2015-05-01

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. 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. 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 setting will have a major role in prioritising malaria control strategies

Prolonged Plasmodium falciparum Infection in Immigrants, Paris

PubMed Central

Godineau, Nadine; Fontanet, Arnaud; Houze, Sandrine; Bouchaud, Olivier; Matheron, Sophie; Le Bras, Jacques

2008-01-01

Few immigrant travelers have Plasmodium falciparum infections >2 months after leaving malaria-endemic areas. We conducted a case–control study to identify factors associated with prolonged P. falciparum infection in immigrant travelers. Results suggest that P. falciparum infection should be systematically suspected, even months after travel, especially in pregnant women and first-arrival immigrants. PMID:18258132

[Isolation and purification of seven catechin compounds from fresh tea leaves by semi-preparative liquid chromatography].

PubMed

Gong, Zhihong; Chen, Si; Gao, Jiangtao; Li, Meihong; Wang, Xiaxia; Lin, Jun; Yu, Xiaomin

2017-11-08

An effective and simple method was established to simultaneously purify seven tea catechins (gallocatechin (GC), epigallocatechin (EGC), catechin (C), epigallocatechin-3- O -gallate (EGCG), epicatechin (EC), epigallocatechin-3- O -(3- O -methyl)-gallate (EGCG3"Me) and epicatechin-3- O -gallate (ECG)) from fresh tea leaves by semi-preparative high performance liquid chromatography (HPLC). Fresh leaves of Tieguanyin tea were successively extracted with methanol and chloroform. Then crude catechins were precipitated from the aqueous fraction of chloroform extraction by adding lead subacetate. Crude catechins were used for the isolation of the seven target catechin compounds by semi-preparative HPLC. Methanol-water and acetonitrile-water were sequentially used as mobile phases. After two rounds of semi-preparative HPLC, all target compounds were achieved with high purities (>90%). The proposed method was tested on two additional tea cultivars and showed similar results. This method demonstrated a simple and efficient strategy based on solvent extraction, ion precipitation and semi-preparative HPLC for the preparation of multiple catechins from tea leaves.

Fresh Osteochondral Allograft Versus Autograft: Twelve-Month Results in Isolated Canine Knee Defects.

PubMed

McCarty, Eric C; Fader, Ryan R; Mitchell, Justin J; Glenn, R Edward; Potter, Hollis G; Spindler, Kurt P

2016-09-01

Osteochondral autografts and allografts have been widely used in the treatment of isolated grade 4 articular cartilage lesions of the knee. However, there is a paucity of literature regarding the basic science investigating the direct comparison between fresh osteochondral allografts to autografts. At 12 months, fresh osteochondral allografts are equal to autografts with respect to function, bony incorporation into host bone, and chondrocyte viability. Controlled laboratory study. Eight adult mongrel dogs underwent bilateral hindlimb osteochondral graft implantation in the knee after creation of an acute Outerbridge grade 4 cartilage defect. One hindlimb of each dog knee received an autograft, and the contralateral knee received an allograft. All dogs were sacrificed at 12 months. Graft analysis included gross examination, radiographs, magnetic resonance imaging (MRI), biomechanical testing, and histology. MRI demonstrated excellent bony incorporation of both autografts and allografts, except for 1 allograft that revealed partial incorporation. Histologic examination of cartilage showed intact hyaline appearance for both autografts and allografts, with fibrocartilage at the host-graft interface of both. Biomechanical testing demonstrated no significant difference between allografts and autografts (P = .76). Furthermore, no significant difference was observed between allografts and the native cartilage with biomechanical testing (P = .84). After 12 months from time of implantation, fresh osteochondral allograft tissue and autograft tissue in this study were not statistically different with respect to biomechanical properties, gross morphology, bony incorporation, or overall histologic characteristics. When compared with the previously reported 6-month incorporation rates, there was improved allograft and autograft incorporation at 12 months. With no significant differences in gross examination, radiographs, MRI, biomechanical testing, or histology in the canine

K13 Propeller Alleles, mdr1 Polymorphism, and Drug Effectiveness at Day 3 after Artemether-Lumefantrine Treatment for Plasmodium falciparum Malaria in Colombia, 2014-2015.

PubMed

Montenegro, Madeline; Neal, Aaron T; Posada, Maritza; De Las Salas, Briegel; Lopera-Mesa, Tatiana M; Fairhurst, Rick M; Tobon-Castaño, Alberto

2017-12-01

High treatment failure rates for Plasmodium falciparum malaria have been reported in Colombia for chloroquine, amodiaquine, and sulfadoxine-pyrimethamine. Artemisinin combination therapies were introduced in 2006 in Colombia, where artemether-lumefantrine (AL) is currently used to treat uncomplicated P. falciparum malaria. Artemisinin (ART) resistance was initially observed in Southeast Asia as an increased parasite clearance time, manifesting as a positive thick-blood smear on day 3 after treatment (D3 positivity). Recently, mutations in the propeller domain of the P. falciparum kelch13 gene ( K13 propeller) have been associated with ART resistance. In this study, we surveyed AL effectiveness at D3 and molecular markers of drug resistance among 187 uncomplicated P. falciparum cases in 4 regions of Colombia from June 2014 to July 2015. We found that 3.2% (4/125) of patients showed D3 positivity, 100% (163/163) of isolates carried wild-type K13 propeller alleles, 12.9% (23/178) of isolates had multiple copies of the multidrug resistance 1 gene ( mdr1 ), and 75.8% (113/149) of isolates harbored the double mutant N F S D D mdr1 haplotype (the underlining indicates mutant alleles). These data suggest that ART resistance is not currently suspected in Colombia but that monitoring for lumefantrine resistance and AL failures should continue. Copyright © 2017 American Society for Microbiology.

Asymptomatic falciparum malaria and intestinal helminths co-infection among school children in Osogbo, Nigeria

PubMed Central

Ojurongbe, Olusola; Adegbayi, Adebola M; Bolaji, Oloyede S; Akindele, Akeem A; Adefioye, Olusegun A; Adeyeba, Oluwaseyi A

2011-01-01

BACKGROUND: Malaria and intestinal helminths are parasitic diseases causing high morbidity and mortality in most tropical parts of the world, where climatic conditions and sanitation practices favor their prevalence. The aim of this study was to determine the prevalence and possible impact of falciparum malaria and intestinal helminths co-infection among school children in Kajola, Osun state, Nigeria. METHODS: Fresh stool and blood samples were collected from 117 primary school children age range 4-15 years. The stool samples were processed using both Kato-Katz and formol-ether concentration techniques and microscopically examined for intestinal parasitic infections. Blood was collected by finger prick to determine malaria parasitemia using thick film method; and packed cell volume (PCV) was determined by hematocrit. Univariate analysis and chi-square statistical tests were used to analyze the data. RESULTS: The prevalence of Plasmodium falciparum, intestinal helminth infections, and co-infection of malaria and helminth in the study were 25.6%, 40.2% and 4.3%, respectively. Five species of intestinal helminths were recovered from the stool samples and these were Ascaris lumbricoides (34.2%), hookworm (5.1%), Trichuris trichiura (2.6%), Diphyllobothrium latum (0.9%) and Trichostrongylus species (0.9%). For the co-infection of both malaria and intestinal helminths, females (5.9%) were more infected than males (2.0%) but the difference was not statistically significant (p = 0.3978). Children who were infected with helminths were equally likely to be infected with malaria as children without intestinal helminths [Risk Ratio (RR) = 0.7295]. Children with A. lumbricoides (RR = 1.359) were also likely to be infected with P. falciparum as compared with uninfected children. CONCLUSIONS: Asymptomatic falciparum malaria and intestinal helminth infections do co-exist without clinical symp-toms in school children in Nigeria. PMID:22091292

Reduced ex vivo susceptibility of Plasmodium falciparum after oral artemether-lumefantrine treatment in Mali.

PubMed

Dama, Souleymane; Niangaly, Hamidou; Ouattara, Amed; Sagara, Issaka; Sissoko, Sekou; Traore, Oumar Bila; Bamadio, Amadou; Dara, Niawanlou; Djimde, Moussa; Alhousseini, Mohamed Lamine; Goita, Siaka; Maiga, Hamma; Dara, Antoine; Doumbo, Ogobara K; Djimde, Abdoulaye A

2017-02-02

Artemisinin-based combination therapy is the recommended first-line treatment for uncomplicated falciparum malaria worldwide. However, recent studies conducted in Mali showed an increased frequency of recurrent parasitaemia following artemether-lumefantrine (AL) treatment. Study samples were collected during a large WANECAM study. Ex-vivo Plasmodium falciparum sensitivity to artemether and lumefantrine was assessed using the tritiated hypoxanthine-based assay. The prevalence of molecular markers of anti-malarial drug resistance (pfcrt K76T, pfmdr1 N86Y and K13-propeller) were measured by PCR and/or sequencing. Overall 61 samples were successfully analysed in ex vivo studies. Mean IC 50 s increased significantly between baseline and recurrent parasites for both artemether (1.6 nM vs 3.2 nM, p < 0.001) and lumefantrine (1.4 nM vs 3.4 nM, p = 0.004). Wild type Pfmdr1 N86 allele was selected after treatment (71 vs 91%, 112 of 158 vs 95 of 105, p < 0.001) but not the wild type pfcrt K76 variant (23.5 vs 24.8%, 40 of 170 vs 26 of 105, p = 0.9). Three non-synonymous K13-propeller SNPs (A522C, A578S, and G638R) were found with allele frequencies <2%. Malian post-AL P. falciparum isolates were less susceptible to artemether and lumefantrine than baseline isolates.

Parasites bearing a single copy of the multi-drug resistance gene (pfmdr-1) with wild-type SNPs predominate amongst Plasmodium falciparum isolates from Malawi.

PubMed

Nkhoma, Standwell; Nair, Shalini; Mukaka, Mavuto; Molyneux, Malcolm E; Ward, Stephen A; Anderson, Timothy J C

2009-07-01

We genotyped 160 P. falciparum infections from Malawi for pfmdr-1 copy number changes and SNPs associated with in vivo tolerance and poor in vitro sensitivity to the component drugs of Coartem. We also measured in vitro susceptibility of 49 of these isolates to a variety of drugs in clinical use or with a potential for use in Africa. All 160 infections carried a single copy of pfmdr-1 but 34% exhibited sequence variation at 4 of the 5 polymorphic sites in pfmdr-1. Isolates carrying 86-Asn and 184-Tyr pfmdr-1 alleles were significantly less sensitive (p<0.001) to mefloquine, lumefantrine, artemether and dihydroartemisinin compared with those bearing 86-Tyr and 184-Phe polymorphisms. This study provides baseline measures prior to policy change: continued surveillance for changes in baseline drug susceptibility, pfmdr-1 copy number and SNPs, and other putative Coartem resistance loci will be necessary to provide an early warning of emerging Coartem resistance in this setting.

Alisiaquinones and alisiaquinol, dual inhibitors of Plasmodium falciparum enzyme targets from a New Caledonian deep water sponge.

PubMed

Desoubzdanne, Denis; Marcourt, Laurence; Raux, Roselyne; Chevalley, Séverine; Dorin, Dominique; Doerig, Christian; Valentin, Alexis; Ausseil, Frédéric; Debitus, Cécile

2008-07-01

Four new meroterpenes, alisiaquinones A-C (1-3) and alisiaquinol (4), were isolated from a New Caledonian deep water sponge. Their structures and relative stereochemistry were elucidated by spectroscopic data analysis. They are related to xestoquinone, but showed unusual substitution on a tetrahydrofuran junction. They displayed micromolar range activity on two enzymatic targets of importance for the control of malaria, the plasmodial kinase Pfnek-1 and a protein farnesyl transferase, as well as on different chloroquine-sensitive and -resistant strains of Plasmodium falciparum. Alisiaquinone C displayed a submicromolar activity on P. falciparum and a competitive selectivity index on the different plasmodial strains.

Novel pfdhps Haplotypes among Imported Cases of Plasmodium falciparum Malaria in the United Kingdom ▿

PubMed Central

Sutherland, Colin J.; Fifer, Helen; Pearce, Richard J.; bin Reza, Faisal; Nicholas, Meredydd; Haustein, Thomas; Njimgye-Tekumafor, Njah E.; Doherty, Justin F.; Gothard, Philip; Polley, Spencer D.; Chiodini, Peter L.

2009-01-01

Treatment of acute malaria caused by Plasmodium falciparum may include long-half-life drugs, such as the antifolate combination sulfadoxine-pyrimethamine (SP), to provide posttreatment chemoprophylaxis against parasite recrudescence or delayed emergence from the liver. An unusual case of P. falciparum recrudescence in a returned British traveler who received such a regimen, as well as a series of 44 parasite isolates from the same hospital, was analyzed by PCR and direct DNA sequencing for the presence of markers of parasite resistance to chloroquine and antifolates. The index patient harbored a mixture of wild-type and resistant pfdhfr and pfdhps alleles upon initial presentation. During his second malaria episode, he harbored only resistant parasites, with the haplotypes IRNI (codons 51, 59, 108, and 164) and SGEAA (codons 436, 437, 540, 581, and 613) at these two loci, respectively. Analysis of isolates from 44 other patients showed that the pfdhfr haplotype IRNI was common (found in 81% of cases). The SGEAA haplotype of pfdhps was uncommon (found only in eight cases of East African origin [17%]). A previously undescribed mutation, I431V, was observed for seven cases of Nigerian origin, occurring as one of two haplotypes, VAGKGS or VAGKAA. The presence of this mutation was also confirmed in isolates of Nigerian origin from the United Kingdom Malaria Reference Laboratory. The presence of the pfdhps haplotype SGEAA in P. falciparum parasites of East African origin appears to compromise the efficacy of treatment regimens that include SP as a means to prevent recrudescence. Parasites with novel pfdhps haplotypes are circulating in West Africa. The response of these parasites to chemotherapy needs to be evaluated. PMID:19433569

Prevalence of Plasmodium falciparum Molecular Markers of Antimalarial Drug Resistance in a Residual Malaria Focus Area in Sabah, Malaysia

PubMed Central

Mohd Abd Razak, Mohd Ridzuan; Abdullah, Noor Rain; Sastu, Umi Rubiah; Imwong, Mallika; Muniandy, Prem Kumar; Saat, Muhammad Nor Farhan; Muhammad, Amirrudin; Jelip, Jenarun; Tikuson, Moizin; Yusof, Norsalleh; Rundi, Christina; Mudin, Rose Nani; Syed Mohamed, Ami Fazlin

2016-01-01

Chloroquine (CQ) and fansidar (sulphadoxine-pyrimethamine, SP) were widely used for treatment of Plasmodium falciparum for several decades in Malaysia prior to the introduction of Artemisinin-based Combination Therapy (ACT) in 2008. Our previous study in Kalabakan, located in south-east coast of Sabah showed a high prevalence of resistance to CQ and SP, suggesting the use of the treatment may no longer be effective in the area. This study aimed to provide a baseline data of antimalarial drug resistant markers on P. falciparum isolates in Kota Marudu located in the north-east coast of Sabah. Mutations on genes associated with CQ (pfcrt and pfmdr1) and SP (pfdhps and pfdhfr) were assessed by PCR amplification and restriction fragment length polymorphism. Mutations on the kelch13 marker (K13) associated with artemisinin resistance were determined by DNA sequencing technique. The assessment of pfmdr1 copy number variation associated with mefloquine resistant was done by real-time PCR technique. A low prevalence (6.9%) was indicated for both pfcrt K76T and pfmdr1 N86Y mutations. All P. falciparum isolates harboured the pfdhps A437G mutation. Prevalence of pfdhfr gene mutations, S108N and I164L, were 100% and 10.3%, respectively. Combining the different resistant markers, only two isolates were conferred to have CQ and SP treatment failure markers as they contained mutant alleles of pfcrt and pfmdr1 together with quintuple pfdhps/pfdhfr mutation (combination of pfdhps A437G+A581G and pfdhfr C59R+S108N+I164L). All P. falciparum isolates carried single copy number of pfmdr1 and wild type K13 marker. This study has demonstrated a low prevalence of CQ and SP resistance alleles in the study area. Continuous monitoring of antimalarial drug efficacy is warranted and the findings provide information for policy makers in ensuring a proper malaria control. PMID:27788228

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

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

Non-falciparum malaria infections in pregnant women in West Africa.

PubMed

Williams, John; Njie, Fanta; Cairns, Matthew; Bojang, Kalifa; Coulibaly, Sheick Oumar; Kayentao, Kassoum; Abubakar, Ismaela; Akor, Francis; Mohammed, Khalifa; Bationo, Richard; Dabira, Edgar; Soulama, Alamissa; Djimdé, Moussa; Guirou, Etienne; Awine, Timothy; Quaye, Stephen L; Ordi, Jaume; Doumbo, Ogobara; Hodgson, Abraham; Oduro, Abraham; Magnussen, Pascal; Ter Kuile, Feiko O; Woukeu, Arouna; Milligan, Paul; Tagbor, Harry; Greenwood, Brian; Chandramohan, Daniel

2016-01-29

Non-Plasmodium falciparum malaria infections are found in many parts of sub-Saharan Africa but little is known about their importance in pregnancy. Blood samples were collected at first antenatal clinic attendance from 2526 women enrolled in a trial of intermittent screening and treatment of malaria in pregnancy (ISTp) versus intermittent preventive treatment (IPTp) conducted in Burkina Faso, The Gambia, Ghana and Mali. DNA was extracted from blood spots and tested for P. falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale using a nested PCR test. Risk factors for a non-falciparum malaria infection were investigated and the influence of these infections on the outcome of pregnancy was determined. P. falciparum infection was detected frequently (overall prevalence by PCR: 38.8 %, [95 % CI 37.0, 40.8]), with a prevalence ranging from 10.8 % in The Gambia to 56.1 % in Ghana. Non-falciparum malaria infections were found only rarely (overall prevalence 1.39 % [95 % CI 1.00, 1.92]), ranging from 0.17 % in the Gambia to 3.81 % in Mali. Ten non-falciparum mono-infections and 25 mixed falciparum and non-falciparum infections were found. P. malariae was the most frequent non-falciparum infection identified; P. vivax was detected only in Mali. Only four of the non-falciparum mono-infections were detected by microscopy or rapid diagnostic test. Recruitment during the late rainy season and low socio-economic status were associated with an increased risk of non-falciparum malaria as well as falciparum malaria. The outcome of pregnancy did not differ between women with a non-falciparum malaria infection and those who were not infected with malaria at first ANC attendance. Non-falciparum infections were infrequent in the populations studied, rarely detected when present as a mono-infection and unlikely to have had an important impact on the outcome of pregnancy in the communities studied due to the small number of women infected with non-falciparum parasites.

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

Two-photon excited autofluorescence imaging of freshly isolated frog retinas.

PubMed

Lu, Rong-Wen; Li, Yi-Chao; Ye, Tong; Strang, Christianne; Keyser, Kent; Curcio, Christine A; Yao, Xin-Cheng

2011-06-01

The purpose of this study was to investigate cellular sources of autofluorescence signals in freshly isolated frog (Rana pipiens) retinas. Equipped with an ultrafast laser, a laser scanning two-photon excitation fluorescence microscope was employed for sub-cellular resolution examination of both sliced and flat-mounted retinas. Two-photon imaging of retinal slices revealed autofluorescence signals over multiple functional layers, including the photoreceptor layer (PRL), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Using flat-mounted retinas, depth-resolved imaging of individual retinal layers further confirmed multiple sources of autofluorescence signals. Cellular structures were clearly observed at the PRL, ONL, INL, and GCL. At the PRL, the autofluorescence was dominantly recorded from the intracellular compartment of the photoreceptors; while mixed intracellular and extracellular autofluorescence signals were observed at the ONL, INL, and GCL. High resolution autofluorescence imaging clearly revealed mosaic organization of rod and cone photoreceptors; and sub-cellular bright autofluorescence spots, which might relate to connecting cilium, was observed in the cone photoreceptors only. Moreover, single-cone and double-cone outer segments could be directly differentiated.

On the Diversity of Malaria Parasites in African Apes and the Origin of Plasmodium falciparum from Bonobos

PubMed Central

Pacheco, M. Andreina; Mugisha, Lawrence; André, Claudine; Halbwax, Michel; Fischer, Anne; Krief, Jean-Michel; Kasenene, John M.; Crandfield, Mike; Cornejo, Omar E.; Chavatte, Jean-Marc; Lin, Clara; Letourneur, Franck; Grüner, Anne Charlotte; McCutchan, Thomas F.; Rénia, Laurent; Snounou, Georges

2010-01-01

The origin of Plasmodium falciparum, the etiological agent of the most dangerous forms of human malaria, remains controversial. Although investigations of homologous parasites in African Apes are crucial to resolve this issue, studies have been restricted to a chimpanzee parasite related to P. falciparum, P. reichenowi, for which a single isolate was available until very recently. Using PCR amplification, we detected Plasmodium parasites in blood samples from 18 of 91 individuals of the genus Pan, including six chimpanzees (three Pan troglodytes troglodytes, three Pan t. schweinfurthii) and twelve bonobos (Pan paniscus). We obtained sequences of the parasites' mitochondrial genomes and/or from two nuclear genes from 14 samples. In addition to P. reichenowi, three other hitherto unknown lineages were found in the chimpanzees. One is related to P. vivax and two to P. falciparum that are likely to belong to distinct species. In the bonobos we found P. falciparum parasites whose mitochondrial genomes indicated that they were distinct from those present in humans, and another parasite lineage related to P. malariae. Phylogenetic analyses based on this diverse set of Plasmodium parasites in African Apes shed new light on the evolutionary history of P. falciparum. The data suggested that P. falciparum did not originate from P. reichenowi of chimpanzees (Pan troglodytes), but rather evolved in bonobos (Pan paniscus), from which it subsequently colonized humans by a host-switch. Finally, our data and that of others indicated that chimpanzees and bonobos maintain malaria parasites, to which humans are susceptible, a factor of some relevance to the renewed efforts to eradicate malaria. PMID:20169187

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

Assessment of antimalarial activity against Plasmodium falciparum and phytochemical screening of some Yemeni medicinal plants

PubMed Central

Alshawsh, Mohammed A.; Al-shamahy, Hassan A.; Alsllami, Salah F.; Lindequist, Ulrike

2009-01-01

Developing countries, where malaria is one of the most prevalent diseases, still rely on traditional medicine as a source for the treatment of this disease. In the present study, six selected plants (Acalypha fruticosa, Azadirachta indica, Cissus rotundifolia, Echium rauwalfii, Dendrosicyos socotrana and Boswellia elongata) commonly used in Yemen by traditional healers for the treatment of malaria as well as other diseases, were collected from different localities of Yemen, dried and extracted with methanol and water successfully. The antiplasmodial activity of the extracts was evaluated against fresh clinical isolates of Plasmodium falciparum. The selectivity parameters to evaluate the efficacy of these medicinal plants were measured by in vitro micro test (Mark III) according to World Health Organization (WHO) 1996 & WHO 2001 protocols of antimalarial drug tests. Among the investigated 12 extracts, three were found to have significant antiplasmodial activity with IC50 values less than 4 µg/ml, namely the water extracts of A. fruticosa, A. indica and D. socotrana. Six extracts showed moderate activity with IC50 values ranging from 10 to 30 µg/ml and three appeared to be inactive with IC50 values more than 30 µg/ml. In addition, preliminary phytochemical screening of the methanolic and aqueous extracts indicated the presence of saponins, tannins, flavonoids, terpenoids, polysaccharides and peptides. PMID:18955251

K13 Propeller Mutations in Plasmodium falciparum Populations in Regions of Malaria Endemicity in Vietnam from 2009 to 2016.

PubMed

Thuy-Nhien, Nguyen; Tuyen, Nguyen Kim; Tong, Nguyen Thanh; Vy, Nguyen Tuong; Thanh, Ngo Viet; Van, Huynh Thuy; Huong-Thu, Pham; Quang, Huynh Hong; Boni, Maciej F; Dolecek, Christiane; Farrar, Jeremy; Thwaites, Guy E; Miotto, Olivo; White, Nicholas J; Hien, Tran Tinh

2017-04-01

The spread of artemisinin-resistant Plasmodium falciparum compromises the therapeutic efficacy of artemisinin combination therapies (ACTs) and is considered the greatest threat to current global initiatives to control and eliminate malaria. This is particularly relevant in Vietnam, where dihydroartemisinin-piperaquine (DP) is the recommended ACT for P. falciparum infection. The propeller domain gene of K13, a molecular marker of artemisinin resistance, was successfully sequenced in 1,060 P. falciparum isolates collected at 3 malaria hot spots in Vietnam between 2009 and 2016. Eight K13 propeller mutations (Thr474Ile, Tyr493His, Arg539Thr, Ile543Thr, Pro553Leu, Val568Gly, Pro574Leu, and Cys580Tyr), including several that have been validated to be artemisinin resistance markers, were found. The prevalences of K13 mutations were 29% (222/767), 6% (11/188), and 43% (45/105) in the Binh Phuoc, Ninh Thuan, and Gia Lai Provinces of Vietnam, respectively. Cys580Tyr became the dominant genotype in recent years, with 79.1% (34/43) of isolates in Binh Phuoc Province and 63% (17/27) of isolates in Gia Lai Province carrying this mutation. K13 mutations were associated with reduced ring-stage susceptibility to dihydroartemisinin (DHA) in vitro and prolonged parasite clearance in vivo An analysis of haplotypes flanking K13 suggested the presence of multiple strains with the Cys580Tyr mutation rather than a single strain expanding across the three sites. Copyright © 2017 Thuy-Nhien et al.

Reduced BRCA1 transcript levels in freshly isolated blood leukocytes from BRCA1 mutation carriers is mutation specific.

PubMed

Chehade, Rania; Pettapiece-Phillips, Rachael; Salmena, Leonardo; Kotlyar, Max; Jurisica, Igor; Narod, Steven A; Akbari, Mohammad R; Kotsopoulos, Joanne

2016-08-17

BRCA1 mutation carriers face a high lifetime risk of developing both breast and ovarian cancer. Haploinsufficiency is thought to predispose these women to cancer by reducing the pool of available BRCA1 transcript and protein, thereby compromising BRCA1 function. Whether or not cancer-free BRCA1 mutation carriers have lower messenger (m)RNA transcript levels in peripheral blood leukocytes has not been evaluated. The primary aim of this study was to characterize an association between BRCA1 mutation status and BRCA1 mRNA leukocyte expression levels among healthy women with a BRCA1 mutation. RNA was extracted from freshly isolated peripheral blood leukocytes of 58 cancer-free, female participants (22 BRCA1 mutation carriers and 36 non-carriers). The expression levels of 236 cancer-associated genes, including BRCA1, were quantified using the Human Cancer Reference gene panel from the Nanostring Technologies nCounter Analysis System. Multivariate modeling demonstrated that carrying a BRCA1 mutation was the most significant predictor of BRCA1 mRNA levels. BRCA1 mRNA levels were significantly lower in BRCA1 mutation carriers compared to non-carriers (146.7 counts vs. 175.1 counts; P = 0.002). Samples with BRCA1 mutations within exon 11 had lower BRCA1 mRNA levels than samples with mutations within the 5' and 3' regions of the BRCA1 gene (122.1 counts vs. 138.9 and 168.6 counts, respectively; P = 0.003). Unsupervised hierarchical clustering of gene expression profiles from freshly isolated blood leukocytes revealed that BRCA1 mutation carriers cluster more closely with other BRCA1 mutation carriers than with BRCA1 wild-type samples. Moreover, a set of 17 genes (including BRCA1) previously shown to be involved in carcinogenesis, were differentially expressed between BRCA1 mutation carriers and non-carriers. Overall, these findings support the concept of BRCA1 haploinsufficiency wherein a specific mutation results in dosage-dependent alteration of BRCA1 at the

Establishment and application of a novel isothermal amplification assay for rapid detection of chloroquine resistance (K76T) in Plasmodium falciparum

PubMed Central

Chahar, Madhvi; Mishra, Neelima; Anvikar, Anup; Dixit, Rajnikant; Valecha, Neena

2017-01-01

Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloroquine resistance transporter (Pfcrt) gene. The point mutation at codon 76 (K76T), which has been observed in more than 91% of P. falciparum isolates in India, is the major determinant of CQ resistance. To overcome the limitations and challenges of traditional methods, in this investigation we developed an easy to use loop mediated isothermal amplification (LAMP) protocol for rapid detection of the K76T mutation associated with CQ resistance in P. falciparum with naked eye visualization. In- house designed primers were synthesized and optimized to specifically distinguish the CQ resistant mutants of P. falciparum. The LAMP reaction was optimal at 61 °C for 60 min and calcein dye was added prior to amplification to enable visual detection. We demonstrate the detection limit of <2 ng/μl respectively, supporting the high sensitivity of this calcein based LAMP method. To the best of our knowledge this is the first report on the establishment of an easy, reliable and cost effective LAMP assay for rapid and specific detection of highly CQ resistance in P. falciparum malaria. PMID:28134241

Isolation, characterization and identification of pericarp-degrading bacteria for the production of off-odour-free white pepper from fresh berries of Piper nigrum L.

PubMed

Vinod, V; Kumar, A; Zachariah, T J

2014-04-01

To isolate, fermentatively evaluate and identify black pepper (Piper nigrum L.)-associated bacteria for the microbial decortication of fresh ripened berries and dried black pepper for preparation of off-odour-free white pepper. Among 45 bacterial isolates obtained from black pepper, seven of them were found to decorticate black pepper (>60%) and fresh pepper berries (98-100%) into white pepper within 5 days of immersion in bacterial suspension. The 16S rRNA genes (1500-bp amplicon) of these bacteria were sequenced, and species identity was established by closest match in GenBank. Superior-quality white pepper was obtained with Bacillus subtilis (IISR WP 33, 34, 38), Bacillus licheniformis (IISR WP 43), Acinetobacter baumanii (IISR WP 35), Klebsiella pneumoniae (IISR WP 19) and Microbacterium barkeri (IISR WP25). The bacterial isolates were found to secrete multiple hydrolytic enzymes such as cellulase, pectinase, amylase, protease and xylanase. Bacterial cultures were deposited with International Depository Authority at Microbial Type Culture Collection, India, as patent deposits as prescribed in Budapest Treaty for microbial deposits. The white pepper, thus obtained from bacterial decortication process, was free from off-odour compound, especially skatole. Other biochemical constituents such as oleoresin, piperine and essential oils were found in the acceptable range. The bacterial decortication did not affect inherent constituents of pepper such as essential oil constituents, oleoresin and piperine content. One of the most significant findings of the work is identification of specific bacterial species for decortication of fresh berries or black pepper berries into value-added white pepper. This work paved way for developing a technological process for microbial decortication of fresh/black pepper for the production of superior-quality white pepper. © 2014 The Society for Applied Microbiology.

Complete genome sequence of Leptospirillum ferrooxidans strain C2-3, isolated from a fresh volcanic ash deposit on the island of Miyake, Japan.

PubMed

Fujimura, Reiko; Sato, Yoshinori; Nishizawa, Tomoyasu; Oshima, Kenshiro; Kim, Seok-Won; Hattori, Masahira; Kamijo, Takashi; Ohta, Hiroyuki

2012-08-01

A diazotrophic, acidophilic, iron-oxidizing bacterium, Leptospirillum ferrooxidans, known to be difficult to cultivate, was isolated from a fresh volcanic ash deposit on the island of Miyake, Japan. Here, we report the complete genome sequence of a cultured strain, C2-3.

Sequence diversity of the C-terminal region of Plasmodium falciparum merozoite surface protein 1 in southern Iran.

PubMed

Zamani, Zahra; Razavi, Mohammad Reza; Sadeghi, Sedigheh; Naddaf, Saeed; Pourfallah, Fatemeh; Mirkhani, Fatemeh; Arjmand, Mohammad; Feizhaddad, Hossein; Rad, Mina Ebrahimi; Ebrahimi Rad, Mina; Tameemi, Marzieh; Assmar, Mehdi

2009-01-01

The C-terminal region of the merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is a strong vaccine candidate as it is associated with immunity to the parasite. This corresponds approximately to the conserved 17th block of the gene and is composed of two EGF- like domains. These domains exhibit only four single amino acid substitutions which show several potential variants in this region of the gene. As the variations might be important for a regional vaccine design, a study was carried out to determine the variations present in P. falciparum isolates from southern Iran. Besides the usual E-T-S-R-L and the Q-K-N-G-F types, we found Q-T-S-R-L, E-K-N-G-F, E-T-S-G-L, Z-T-S-G-L and Z-T-S-R-L types, where Z was E or Q signifying the presence of mixed clones in single isolates.

Mosquito Vectors and the Globalization of Plasmodium falciparum Malaria.

PubMed

Molina-Cruz, Alvaro; Zilversmit, Martine M; Neafsey, Daniel E; Hartl, Daniel L; Barillas-Mury, Carolina

2016-11-23

Plasmodium falciparum malaria remains a devastating public health problem. Recent discoveries have shed light on the origin and evolution of Plasmodium parasites and their interactions with their vertebrate and mosquito hosts. P. falciparum malaria originated in Africa from a single horizontal transfer between an infected gorilla and a human, and became global as the result of human migration. Today, P. falciparum malaria is transmitted worldwide by more than 70 different anopheline mosquito species. Recent studies indicate that the mosquito immune system can be a barrier to malaria transmission and that the P. falciparum Pfs47 gene allows the parasite to evade mosquito immune detection. Here, we review the origin and globalization of P. falciparum and integrate this history with analysis of the biology, evolution, and dispersal of the main mosquito vectors. This new perspective broadens our understanding of P. falciparum population structure and the dispersal of important parasite genetic traits.

Drug Evaluation in the Plasmodium Falciparum-Aotus Model.

DTIC Science & Technology

1986-09-30

strains of Plasmodium falciparum, Uganda Palo Alto ( chloroquine sensi- tive) or Vietnam Smith (chioroquine resistant), in Aotus trivirgatus, were used...Plasmodium falciparum in the Panamanian owl monkey Aotus Two strains of falciparum malaria, Uganda Palo Alto (sensitive to chloroquine anfd quinine...resistant to pyrimetha- mine) and Vietnam Smith (resistant to chloroquine , quinine and pyrimethamine) were used. Previous evaluation of two stereoisomers of

Isolation and characterisation of an enterocin P-producing Enterococcus lactis strain from a fresh shrimp (Penaeus vannamei).

PubMed

Ben Braïek, Olfa; Ghomrassi, Hamdi; Cremonesi, Paola; Morandi, Stefano; Fleury, Yannick; Le Chevalier, Patrick; Hani, Khaled; Bel Hadj, Omrane; Ghrairi, Taoufik

2017-06-01

Screening for lactic acid bacteria (LAB) from fresh shrimp samples (Penaeus vannamei) collected from retail seafood markets in the Tunisian's coast, resulted in the isolation of an Enterococcus strain termed Q1. This strain was selected for its antagonistic activity against pathogenic bacteria such as Listeria monocytogenes, Pseudomonas aeruginosa, Lactococcus garvieae and against fungi (Aspergillus niger and Fusarium equiseti). The Q1 strain was characterised using standard morphological and biochemical tests, growth assays at different temperatures, pH and salinity. 16S rRNA, rpoA and pheS gene sequencing, as well as the 16S-23S rRNA intergenic spacer analyses, were combined to identify strain Q1 as a strain of Enterococcus lactis. The bacteriocin produced by E. lactis Q1 is thermostable, active in the pH range from 4.0 to 9.0 and has a bactericidal mode of action. The enterocin P structural gene was detected by specific PCR in strain E. lactis Q1, which is in good agreement with SDS-PAGE data of the purified bacteriocin. A lack of significant antibiotic resistance genes and virulence determinants was confirmed by specific PCRs. This work provides the first description of an enterocin P producer E. lactis strain isolated from a fresh shrimp. Based on its safety properties (absence of haemolytic activity, virulence factors and antibiotic resistance genes), this strain has the potential to be used as a natural additive or adjunct protective culture in food biopreservation and/or probiotic culture.

A histidine-rich protein 2-based malaria drug sensitivity assay for field use.

PubMed

Noedl, Harald; Attlmayr, Bernhard; Wernsdorfer, Walther H; Kollaritsch, Herwig; Miller, Robert S

2004-12-01

With the spread of antimalarial drug resistance, simple and reliable tools for the assessment of antimalarial drug resistance, particularly in endemic regions and under field conditions, have become more important than ever before. We therefore developed a histidine-rich protein 2 (HRP2)-based drug sensitivity assay for testing of fresh isolates of Plasmodium falciparum in the field. In contrast to the HRP2 laboratory assay, the field assay uses a procedure that further simplifies the handling and culturing of malaria parasites by omitting centrifugation, washing, the use of serum, and dilution with uninfected red blood cells. A total of 40 fresh Plasmodium falciparum isolates were successfully tested for their susceptibility to dihydroartemisinin, mefloquine, quinine, and chloroquine (50% inhibitory concentration [IC50] = 3.43, 61.89, 326.75, and 185.31 nM, respectively). Results very closely matched those obtained with a modified World Health Organization schizont maturation assay (R2 = 0.96, P < 0.001; mean log difference at IC50 = 0.054).

Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

PubMed Central

Ostera, Graciela; Tokumasu, Fuyuki; Oliveira, Fabiano; Sa, Juliana; Furuya, Tetsuya; Teixeira, Clarissa; Dvorak, James

2008-01-01

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P.falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite, PMID:18504040

Complete Genome Sequence of Leptospirillum ferrooxidans Strain C2-3, Isolated from a Fresh Volcanic Ash Deposit on the Island of Miyake, Japan

PubMed Central

Fujimura, Reiko; Sato, Yoshinori; Nishizawa, Tomoyasu; Oshima, Kenshiro; Kim, Seok-Won; Hattori, Masahira; Kamijo, Takashi

2012-01-01

A diazotrophic, acidophilic, iron-oxidizing bacterium, Leptospirillum ferrooxidans, known to be difficult to cultivate, was isolated from a fresh volcanic ash deposit on the island of Miyake, Japan. Here, we report the complete genome sequence of a cultured strain, C2-3. PMID:22815442

Cytoadherence and Genotype of Plasmodium falciparum Strains from Symptomatic Children in Franceville, Southeastern Gabon

PubMed Central

Touré, Fousseyni S.; Ouwe-Missi-Oukem-Boyer, Odile; Mezui-Me-Ndong, Jérôme; Ndong-Atome, Guy Roger; Bisvigou, Ulrick; Mazier, Dominique; Bisser, Sylvie

2007-01-01

Background: Plasmodium falciparum causes severe clinical manifestations by sequestering parasitized red blood cells (PRBC) in the microvasculature of major organs such as the brain. This sequestration results from PRBC adherence to vascular endothelial cells via erythrocyte membrane protein 1, a variant parasite surface antigen. Objective: To determine whether P. falciparum multiple genotype infection (MGI) is associated with stronger PRBC cytoadherence and greater clinical severity. Methods: Nested polymerase chain reaction was used to genotype P. falciparum isolates from symptomatic children and to distinguish between single genotype infection (SGI) and MGI. PRBC cytoadhesion was studied with cultured human lung endothelial cells. Results: Analysis of two highly polymorphic regions of the merozoite surface antigen (MSP)-1 and MSP-2 genes and a dimorphic region of the erythrocyte binding antigen-175 gene showed that 21.4% and 78.6% of the 42 children had SGI and MGI, respectively. It also showed that 37 (89%) of the 42 PRBC samples expressed MSP-1 allelic family K1. Cytoadherence values ranged from 58 to 1811 PRBC/mm2 of human lung endothelial cells monolayer in SGI and from 5 to 5744 PRBC/mm2 in MGI. MGI was not associated with higher cytoadherence values or with more severe malaria. Conclusions: These results suggested that infection of the same individual by multiple clones of P. falciparum does not significantly influence PRBC cytoadherence or disease severity and confirmed the predominance of the MSP-1 K1 genotype in southeastern Gabon. PMID:17607045

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

Diagnosis of Plasmodium falciparum infection in man: detection of parasite antigens by ELISA*

PubMed Central

Mackey, L. J.; McGregor, I. A.; Paounova, N.; Lambert, P. H.

1982-01-01

An ELISA method has been developed for the diagnosis of Plasmodium falciparum infection in man. Parasites from in vitro cultures of P. falciparum were used as source of antigen for the solid phase and the source of specific antibody was immune Gambian sera; binding of antibody in antigen-coated wells was registered by means of alkaline phosphatase-conjugated anti-human IgG. Parasites were detected on the basis of inhibition of antibody-binding. The test was applied to the detection of parasites in human red blood cells (RBC) from in vitro cultures of P. falciparum and in RBC from infected Gambians; RBC from 100 Geneva blood donors served as normal, uninfected controls. In titration experiments, the degree of antibody-binding inhibition correlated with the number of parasites in the test RBC. Parasites were detected at a level of 8 parasites/106 RBC. Samples of RBC were tested from 126 Gambians with microscopically proven infection; significant antibody-binding inhibition was found in 86% of these cases, where parasitaemia ranged from 10 to 125 000/μl of blood. The presence of high-titre antibody in the test preparations was found to reduce the sensitivity of parasite detection in infected RBC from in vitro cultures mixed with equal volumes of different antibody-containing sera. The sensitivity was restored in most cases by recovering the RBC by centrifugation before testing. In a preliminary experiment, there was no significant difference in antibody-binding inhibition using fresh infected RBC and RBC dried on filter-paper and recovered by elution, although there was greater variation in the latter samples. PMID:7044589

Unraveling Haplotype Diversity of the Apical Membrane Antigen-1 Gene in Plasmodium falciparum Populations in Thailand

PubMed Central

Lumkul, Lalita; Sawaswong, Vorthon; Simpalipan, Phumin; Kaewthamasorn, Morakot; Harnyuttanakorn, Pongchai; Pattaradilokrat, Sittiporn

2018-01-01

Development of an effective vaccine is critically needed for the prevention of malaria. One of the key antigens for malaria vaccines is the apical membrane antigen 1 (AMA-1) of the human malaria parasite Plasmodium falciparum, the surface protein for erythrocyte invasion of the parasite. The gene encoding AMA-1 has been sequenced from populations of P. falciparum worldwide, but the haplotype diversity of the gene in P. falciparum populations in the Greater Mekong Subregion (GMS), including Thailand, remains to be characterized. In the present study, the AMA-1 gene was PCR amplified and sequenced from the genomic DNA of 65 P. falciparum isolates from 5 endemic areas in Thailand. The nearly full-length 1,848 nucleotide sequence of AMA-1 was subjected to molecular analyses, including nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity and neutrality tests. Phylogenetic analysis and pairwise population differentiation (Fst indices) were performed to infer the population structure. The analyses identified 60 single nucleotide polymorphic loci, predominately located in domain I of AMA-1. A total of 31 unique AMA-1 haplotypes were identified, which included 11 novel ones. The phylogenetic tree of the AMA-1 haplotypes revealed multiple clades of AMA-1, each of which contained parasites of multiple geographical origins, consistent with the Fst indices indicating genetic homogeneity or gene flow among geographically distinct populations of P. falciparum in Thailand’s borders with Myanmar, Laos and Cambodia. In summary, the study revealed novel haplotypes and population structure needed for the further advancement of AMA-1-based malaria vaccines in the GMS. PMID:29742870

Antimalarial Activities of Peptide Antibiotics Isolated from Fungi

PubMed Central

Nagaraj, G.; Uma, M. V.; Shivayogi, M. S.; Balaram, Hemalatha

2001-01-01

Malaria caused by Plasmodium falciparum is a major public health problem in the developing countries of the world. Clinical treatment of malaria has become complicated due to the occurrence of infections caused by drug resistant parasites. Secondary metabolites from fungi are an attractive source of chemotherapeutic agents. This work reports the isolation and in vitro antiplasmodial activities of peptide antibiotics of fungal origin. The three peptide antibiotics used in this study were efrapeptins, zervamicins, and antiamoebin. The high-performance liquid chromatography-purified peptides were characterized by nuclear magnetic resonance and mass spectral analysis. All three fungal peptides kill P. falciparum in culture with 50% inhibitory concentrations in the micromolar range. A possible mode of action of these peptide antibiotics on P. falciparum is presented. PMID:11120957

K13 mutations and pfmdr1 copy number variation in Plasmodium falciparum malaria in Myanmar.

PubMed

Win, Aye A; Imwong, Mallika; Kyaw, Myat P; Woodrow, Charles J; Chotivanich, Kesinee; Hanboonkunupakarn, Borimas; Pukrittayakamee, Sasithon

2016-02-24

Artemisinin-based combination therapy has been first-line treatment for falciparum malaria in Myanmar since 2005. The wide extent of artemisinin resistance in the Greater Mekong sub-region and the presence of mefloquine resistance at the Myanmar-Thailand border raise concerns over resistance patterns in Myanmar. The availability of molecular markers for resistance to both drugs enables assessment even in remote malaria-endemic areas. A total of 250 dried blood spot samples collected from patients with Plasmodium falciparum malarial infection in five malaria-endemic areas across Myanmar were analysed for kelch 13 sequence (k13) and pfmdr1 copy number variation. K13 mutations in the region corresponding to amino acids 210-726 (including the propeller region of the protein) were detected by nested PCR amplification and sequencing, and pfmdr1 copy number variation by real-time PCR. In two sites, a sub-set of patients were prospectively followed up for assessment of day-3 parasite clearance rates after a standard course of artemether-lumefantrine. K13 mutations and pfmdr1 amplification were successfully analysed in 206 and 218 samples, respectively. Sixty-nine isolates (33.5 %) had mutations within the k13 propeller region with 53 of these (76.8 %) having mutations already known to be associated with artemisinin resistance. F446I (32 isolates) and P574L (15 isolates) were the most common examples. K13 mutation was less common in sites in western border regions (29 of 155 isolates) compared to samples from the east and north (40 of 51 isolates; p < 0.0001). The overall proportion of parasites with multiple pfmdr1 copies (greater than 1.5) was 5.5 %. Seven samples showed both k13 mutation and multiple copies of pfmdr1. Only one of 36 patients followed up after artemether-lumefantrine treatment still had parasites at day 3; molecular analysis indicated wild-type k13 and single copy pfmdr1. The proportion of P. falciparum isolates with mutations in the propeller region of k

Prevalence of K13-propeller gene polymorphisms among Plasmodium falciparum parasites isolated from adult symptomatic patients in northern Uganda.

PubMed

Ocan, Moses; Bwanga, Freddie; Okeng, Alfred; Katabazi, Fred; Kigozi, Edgar; Kyobe, Samuel; Ogwal-Okeng, Jasper; Obua, Celestino

2016-08-19

In the absence of an effective vaccine, malaria treatment and eradication is still a challenge in most endemic areas globally. This is especially the case with the current reported emergence of resistance to artemisinin agents in Southeast Asia. This study therefore explored the prevalence of K13-propeller gene polymorphisms among Plasmodium falciparum parasites in northern Uganda. Adult patients (≥18 years) presenting to out-patients department of Lira and Gulu regional referral hospitals in northern Uganda were randomly recruited. Laboratory investigation for presence of plasmodium infection among patients was done using Plasmodium falciparum exclusive rapid diagnostic test, histidine rich protein-2 (HRP2) (Pf). Finger prick capillary blood from patients with a positive malaria test was spotted on a filter paper Whatman no. 903. The parasite DNA was extracted using chelex resin method and sequenced for mutations in K13-propeller gene using Sanger sequencing. PCR DNA sequence products were analyzed using in DNAsp 5.10.01software, data was further processed in Excel spreadsheet 2007. A total of 60 parasite DNA samples were sequenced. Polymorphisms in the K13-propeller gene were detected in four (4) of the 60 parasite DNA samples sequenced. A non-synonymous polymorphism at codon 533 previously detected in Cambodia was found in the parasite DNA samples analyzed. Polymorphisms at codon 522 (non-synonymous) and codon 509 (synonymous) were also found in the samples analyzed. The study found evidence of positive selection in the Plasmodium falciparum population in northern Uganda (Tajima's D = -1.83205; Fu and Li's D = -1.82458). Polymorphism in the K13-propeller gene previously reported in Cambodia has been found in the Ugandan Plasmodium falciparum parasites. There is need for continuous surveillance for artemisinin resistance gene markers in the country.

Clinical trial of extended-dose chloroquine for treatment of resistant falciparum malaria among Afghan refugees in Pakistan.

PubMed

Howard, Natasha; Durrani, Naeem; Sanda, Sanda; Beshir, Khalid; Hallett, Rachel; Rowland, Mark

2011-06-23

Falciparum malaria is a significant problem for Afghan refugees in Pakistan. Refugee treatment guidelines recommended standard three-day chloroquine treatment (25 mg/kg) for first episodes and extended five-day treatment (40 mg/kg) for recrudescent infections, based on the assumption that a five-day course would more likely achieve a cure. An in-vivo randomized controlled trial was conducted among refugees with uncomplicated falciparum malaria to determine whether five-day treatment (CQ40) was more effective than standard treatment (CQ25). 142 falciparum patients were recruited into CQ25 or CQ40 treatment arms and followed up to 60 days with regular blood smears. The primary outcome was parasitological cure without recrudescence. Treatment failures were retreated with CQ40. PCR genotyping of 270 samples, from the same and nearby sites, was used to support interpretation of outcomes. 84% of CQ25 versus 51% of CQ40 patients experienced parasite recrudescence during follow-up (adjusted odds ratio 0.17, 95%CI 0.08-0.38). Cure rates were significantly improved with CQ40, particularly among adults. Fever clearance time, parasite clearance time, and proportions gametocytaemic post-treatment were similar between treatment groups. Second-line CQ40 treatment resulted in higher failure rates than first-line CQ40 treatment. CQ-resistance marker pfcrt 76T was found in all isolates analysed, while pfmdr1 86Y and 184Y were found in 18% and 37% of isolates respectively. CQ is not suitable for first-line falciparum treatment in Afghan refugee communities. The extended-dose CQ regimen can overcome 39% of resistant infections that would recrudesce under the standard regimen, but the high failure rate after directly observed treatment demonstrates its use is inappropriate.

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

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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

PubMed

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

2016-04-01

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). 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. 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). 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).

Molecular Epidemiology of Malaria in Cameroon and Côte d'Ivoire. XXXI. Kelch 13 Propeller Sequences in Plasmodium falciparum Isolates before and after Implementation of Artemisinin-Based Combination Therapy.

PubMed

Djaman, Joseph Allico; Olefongo, Dagnogo; Ako, Aristide Berenger; Roman, Jocelyne; Ngane, Vincent Foumane; Basco, Leonardo K; Tahar, Rachida

2017-07-01

Artemisinin-resistant malaria has not been reported from Africa, but resistance can possibly spread from Asia or arise independently in Africa. The emergence of artemisinin resistance in Africa can be monitored by molecular assay of Kelch 13 (K13) propeller sequences. A total of 251 archived DNA samples of Plasmodium falciparum isolates collected in 2002, 2003, and 2006 in Yaounde, Cameroon, and 47 samples collected in 2006 and 2013 in Abidjan, Côte d'Ivoire, were analyzed for K13-propeller sequence polymorphism. Only one isolate carried a mutant K13-propeller allele (E602D). None of the isolates carried the key mutant alleles (Y493H, R539T, I543T, and C580Y) associated with artemisinin resistance in Cambodia. The presence of the mutant allele was not correlated with in vitro response to dihydroartemisinin determined by the classical hypoxanthine incorporation assay. There was no evidence of K13 mutations associated with artemisinin resistance before and soon after the introduction of artemisinin-based combination therapies in Cameroon and Côte d'Ivoire.

Antiplasmodial activity of flavonol quercetin and its analogues in Plasmodium falciparum: evidence from clinical isolates in Bangladesh and standardized parasite clones.

PubMed

Ganesh, Deepa; Fuehrer, Hans-Peter; Starzengrüber, Peter; Swoboda, Paul; Khan, Wasif Ali; Reismann, Johannes A B; Mueller, Milena S K; Chiba, Peter; Noedl, Harald

2012-06-01

Malaria is still a major threat in many parts of the world with resistance spreading to almost all classes of antimalarials. The limited arsenal of available antimalarial drugs emphasizes the urgent need for novel antimalarial compounds. Owing to the fact that novel leads from nature have traditionally played a pivotal role in the development of various classes of antimalarials, we investigated a set of eight naturally occurring dietary flavonoids and their analogues for their antiplasmodial activity on clinical field isolates in southeastern Bangladesh and culture-adapted chloroquine-sensitive and chloroquine-resistant parasite clones. Except for taxifolin, all the other flavonoids had 50% inhibitory concentrations below 14 μM, both in the field and laboratory-adapted parasites. Neither of the flavonoids showed any activity correlation with chloroquine. The quercetin analogue rutin (7.10 ± 10.32 μM) was the most active substance in field isolates as well as laboratory-adapted cultures (3.53 ± 13.34 μM in 3D7 and 10.38 ± 15.08 μM in K1), providing the first evidence of its activity against Plasmodium falciparum parasites. Thus, our results provide important evidence of the antimalarial activity of flavonoids in traditional use and thus warrant further investigation of these compounds as potential antiplasmodial agents.

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

2016-10-03

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetic mutations in Plasmodium falciparum and Plasmodium vivax dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) in Vanuatu and Solomon Islands prior to the introduction of artemisinin combination therapy.

PubMed

Gresty, Karryn J; Gray, Karen-Ann; Bobogare, Albino; Wini, Lyndes; Taleo, George; Hii, Jeffrey; Cheng, Qin; Waters, Norman C

2014-10-14

Plasmodium falciparum and Plasmodium vivax are endemic in Vanuatu and the Solomon Islands. While both countries have introduced artemether-lumefantrine (AL) as first-line therapy for both P. falciparum and P. vivax since 2008, chloroquine and sulphadoxine-pyrimethamine (SP) were used as first-line therapy for many years prior to the introduction of AL. Limited data are available on the extent of SP resistance at the time of policy change. Blood spots were obtained from epidemiological surveys conducted on Tanna Island, Tafea Province, Vanuatu and Temotu Province, Solomon Islands in 2008. Additional samples from Malaita Province, Solomon Islands were collected as part of an AL therapeutic efficacy study conducted in 2008. Plasmodium vivax and P. falciparum dhfr and dhps genes were sequenced to detect nucleotide polymorphisms. All P. falciparum samples analysed (n=114) possessed a double mutant pfdhfr allele (C59R/S108N). Additionally, mutation A437G in pfhdps was detected in a small number of samples 2/13, 1/17 and 3/26 from Tanna Island, Vanuatu and Temotu and Malaita Provinces Solomon Islands respectively. Mutations were also common in pvdhfr from Tanna Island, Vanuatu, where 33/51 parasites carried the double amino acid substitution S58R/S117N, while in Temotu and Malaita Provinces, Solomon Islands 32/40 and 39/46 isolates carried the quadruple amino acid substitution F57L/S58R/T61M/S117T in DHFR respectively. No mutations in pvdhps (n=108) were detected in these three island groups. Prior to the introduction of AL, there was a moderate level of SP resistance in the P. falciparum population that may cause SP treatment failure in young children. Of the P. vivax isolates, a majority of Solomon Islands isolates carried quadruple mutant pvdhfr alleles while a majority of Vanuatu isolates carried double mutant pvdhfr alleles. This suggests a higher level of SP resistance in the P. vivax population in Solomon Islands compared to the sympatric P. falciparum population

Submicroscopic placental infection by non-falciparum Plasmodium spp.

PubMed Central

Doritchamou, Justin Y. A.; Akuffo, Richard A.; Moussiliou, Azizath; Luty, Adrian J. F.; Massougbodji, Achille; Deloron, Philippe

2018-01-01

Background Among the Plasmodium species that infect humans, adverse effects of P. falciparum and P. vivax have been extensively studied and reported with respect to poor outcomes particularly in first time mothers and in pregnant women living in areas with unstable malaria transmission. Although, other non-falciparum malaria infections during pregnancy have sometimes been reported, little is known about the dynamics of these infections during pregnancy. Methods and findings Using a quantitative PCR approach, blood samples collected from Beninese pregnant women during the first antenatal visit (ANV) and at delivery including placental blood were screened for Plasmodium spp. Risk factors associated with Plasmodium spp. infection during pregnancy were assessed as well as the relationships with pregnancy outcomes. P. falciparum was the most prevalent Plasmodium species detected during pregnancy, irrespective either of parity, of age or of season during which the infection occurred. Although no P. vivax infections were detected in this cohort, P. malariae (9.2%) and P. ovale (5.8%) infections were observed in samples collected during the first ANV. These non-falciparum infections were also detected in maternal peripheral blood (1.3% for P. malariae and 1.2% for P. ovale) at delivery. Importantly, higher prevalence of P. malariae (5.5%) was observed in placental than peripheral blood while that of P. ovale was similar (1.8% in placental blood). Among the non-falciparum infected pregnant women with paired peripheral and placental samples, P. malariae infections in the placental blood was significantly higher than in the peripheral blood, suggesting a possible affinity of P. malariae for the placenta. However, no assoctiation of non-falciparum infections and the pregnancy outcomes was observed Conclusions Overall this study provided insights into the molecular epidemiology of Plasmodium spp. infection during pregnancy, indicating placental infection by non-falciparum

Submicroscopic placental infection by non-falciparum Plasmodium spp.

PubMed

Doritchamou, Justin Y A; Akuffo, Richard A; Moussiliou, Azizath; Luty, Adrian J F; Massougbodji, Achille; Deloron, Philippe; Tuikue Ndam, Nicaise G

2018-02-01

Among the Plasmodium species that infect humans, adverse effects of P. falciparum and P. vivax have been extensively studied and reported with respect to poor outcomes particularly in first time mothers and in pregnant women living in areas with unstable malaria transmission. Although, other non-falciparum malaria infections during pregnancy have sometimes been reported, little is known about the dynamics of these infections during pregnancy. Using a quantitative PCR approach, blood samples collected from Beninese pregnant women during the first antenatal visit (ANV) and at delivery including placental blood were screened for Plasmodium spp. Risk factors associated with Plasmodium spp. infection during pregnancy were assessed as well as the relationships with pregnancy outcomes. P. falciparum was the most prevalent Plasmodium species detected during pregnancy, irrespective either of parity, of age or of season during which the infection occurred. Although no P. vivax infections were detected in this cohort, P. malariae (9.2%) and P. ovale (5.8%) infections were observed in samples collected during the first ANV. These non-falciparum infections were also detected in maternal peripheral blood (1.3% for P. malariae and 1.2% for P. ovale) at delivery. Importantly, higher prevalence of P. malariae (5.5%) was observed in placental than peripheral blood while that of P. ovale was similar (1.8% in placental blood). Among the non-falciparum infected pregnant women with paired peripheral and placental samples, P. malariae infections in the placental blood was significantly higher than in the peripheral blood, suggesting a possible affinity of P. malariae for the placenta. However, no assoctiation of non-falciparum infections and the pregnancy outcomes was observed. Overall this study provided insights into the molecular epidemiology of Plasmodium spp. infection during pregnancy, indicating placental infection by non-falciparum Plasmodium and the lack of association of these

High performance of histidine-rich protein 2 based rapid diagnostic tests in French Guiana are explained by the absence of pfhrp2 gene deletion in P. falciparum.

PubMed

Trouvay, Mélanie; Palazon, Georges; Berger, Franck; Volney, Béatrice; Blanchet, Denis; Faway, Emilie; Donato, Damien; Legrand, Eric; Carme, Bernard; Musset, Lise

2013-01-01

Care for malaria patients in endemic areas has been improved through the increasing use of Rapid Diagnostic Tests (RDTs). Most RDTs target the histidine-rich protein-2 antigen (PfHRP2) to detect P. falciparum, as it is abundant and shows great heat stability. However, their use in South America has been widely questioned following a recent publication that pinpoints the high prevalence of Peruvian field isolates lacking the gene encoding this protein. In the remote rural health centers of French Guiana, RDTs are the main diagnosis tools. Therefore, a study of PfHRP2 RDT performances and pfhrp2 genotyping was conducted to determine whether a replacement of the current pLDH-based kit could be considered. The performance study compared the SD Malaria Ag test P.f/Pan® kit with the current gold standard diagnosis by microscopy. The prevalence of pfhrp2 and pfhrp3 deletions were evaluated from 221 P. falciparum isolates collected between 2009 and 2011 in French Guiana. Between January 2010 and August 2011, 960 suspected cases of malaria were analyzed using microscopy and RDTs. The sensitivity of the SD Malaria Ag test P.f/Pan® for detection of P. falciparum was 96.8% (95% CI: 90.9-99.3), and 86.0% (95% CI: 78.9-91.5) for the detection of P. vivax. No isolates (95% CI: 0-4.5) lacking either exon of the pfhrp2 gene were identified among the 221 P. falciparum isolates analyzed, but 7.4% (95% CI: 2.8-15.4) lacked the exon 2 part of the pfhrp3 gene. Field isolates lacking either exon of the pfhrp2 gene are absent in this western part of South America. Despite its sensibility to detect P. vivax, the SD Malaria Ag test P.f/Pan® kit is a satisfying alternative to microscopy in remote health centers, where it is difficult to provide highly skilled microscopists and to maintain the necessary equipment.

Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum.

PubMed

El Bissati, Kamal; Downie, Megan J; Kim, Seong-Kyoun; Horowitz, Michael; Carter, Nicola; Ullman, Buddy; Ben Mamoun, Choukri

2008-10-01

The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine.

Development of a potential probiotic fresh cheese using two Lactobacillus salivarius strains isolated from human milk.

PubMed

Cárdenas, Nivia; Calzada, Javier; Peirotén, Angela; Jiménez, Esther; Escudero, Rosa; Rodríguez, Juan M; Medina, Margarita; 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.

N sub 2 -fixation by freshly isolated Nostoc from coralloid roots of the cycad Macrozamia riedlei (Fisch. ex Gaud. ) Gardn

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lindblad, P.; Atkins, C.A.; Pate, J.S.

1991-03-01

Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation ({sup 15}N{sub 2} fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. The data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O{sub 2}, and a direct dependence on the cycad for substrates to support nitrogenase activity.

Plasmodium falciparum population structure in Sudan post artemisinin-based combination therapy.

PubMed

Bakhiet, Amani M A; Abdel-Muhsin, Abdel-Muhsin A; Elzaki, Salah-Eldin G; Al-Hashami, Zainab; Albarwani, Hamida S; AlQamashoui, Badar A; Al-Hamidhi, Salama; Idris, Mohamed A; Elagib, Atif A; Beja-Pereira, Albano; Babiker, Hamza A

2015-08-01

Over the past decade, Sudan has stepped up malaria control backed by WHO, and this has resulted in significant reduction in parasite rate, malaria morbidity and mortality. The present study analyzed Plasmodium falciparum parasites in four geographical separated areas, to examine whether the success in malaria control following the use of artemisinin-based combination therapy (ACT) has disrupted the population structure and evolution of the parasite. We examined 319 P. falciparum isolates collected between October 2009 and October 2012 in four different areas in Sudan (Jazira [central Sudan], Southern Darfur [western Sudan], Upper Nile [southern Sudan] and Kasala [eastern Sudan]). Twelve microsatellites were analyzed for allelic diversity, multi-locus haplotype and inter-population differentiation. Level of diversity was compared to that detected for three of the above microsatellites among P. falciparum parasites in central and eastern Sudan in 1999, prior to introduction of ACT. Diversity at each locus (unbiased heterozygosity [H]) was high in all areas (Jazira, H=0.67), (Southern Darfur, H=0.71), (Upper Nile, H=0.71), and (Kasala, H=0.63). Microsatellites were distributed widely and private alleles, detected in a single population, were rare. The extent of diversity in the above sites was similar to that seen, in 1999, in central (Khartoum, H=0.73) and eastern Sudan (Gedaref, H=0.75). Significant Linkage disequilibrium (LD) was observed between the microsatellites in all populations. Pairwise FST analysis revealed that parasites in the four areas could be considered as one population. However, the parasites in Sudan clustered away from parasites in West Africa and the Arabian Peninsula. Despite marked reduction in malaria risk in Sudan, the extent of diversity and parasite genetic structure are indicative of a large population size. Further considerable reduction in transmission would be needed before fragmented sub-population can be seen. In addition, the large

Transfer and detection of freshly isolated or cultured chicken (Gallus gallus) and exotic species' embryonic gonadal germ stem cells in host embryos.

PubMed

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

2014-06-01

The management of captive avian breeding programs increasingly utilizes various artificial reproductive technologies, including in ovo 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.

Origin and Dissemination of Chloroquine-Resistant Plasmodium falciparum with Mutant pfcrt Alleles in the Philippines

PubMed Central

Chen, Nanhua; Wilson, Danny W.; Pasay, Cielo; Bell, David; Martin, Laura B.; Kyle, Dennis; Cheng, Qin

2005-01-01

The pfcrt allelic type and adjacent microsatellite marker type were determined for 82 Plasmodium falciparum isolates from the Philippines. Mutant pfcrt allelic types P1a and P2a/P2b were dominant in different locations. Microsatellite analysis revealed that P2a/P2b evolved independently in the Philippines, while P1a shared common ancestry with Papua New Guinea chloroquine-resistant parasites. PMID:15855538

Genetic diversity in the merozoite surface protein 1 and 2 genes of Plasmodium falciparum from the Artibonite Valley of Haiti.

PubMed

Londono-Renteria, Berlin; Eisele, Thomas P; Keating, Joseph; Bennett, Adam; Krogstad, Donald J

2012-01-01

Describing genetic diversity of the Plasmodium falciparum parasite provides important information about the local epidemiology of malaria. In this study, we examined the genetic diversity of P. falciparum isolates from the Artibonite Valley in Haiti using the allelic families of merozoite surface protein 1 and 2 genes (msp-1 and msp-2). The majority of study subjects infected with P. falciparum had a single parasite genotype (56% for msp-1 and 69% for msp-2: n=79); 9 distinct msp-1 genotypes were identified by size differences on agarose gels. K1 was the most polymorphic allelic family with 5 genotypes (amplicons from 100 to 300 base pairs [bp]); RO33 was the least polymorphic, with a single genotype (120-bp). Although both msp-2 alleles (3D7/IC1, FC27) had similar number of genotypes (n=4), 3D7/IC1 was more frequent (85% vs. 26%). All samples were screened for the presence of the K76T mutation on the P. falciparum chloroquine resistance transporter (pfcrt) gene with 10 of 79 samples positive. Of the 2 (out of 10) samples from individuals follow-up for 21 days, P. falciparum parasites were present through day 7 after treatment with chloroquine. No parasites were found on day 21. Our results suggest that the level of genetic diversity is low in this area of Haiti, which is consistent with an area of low transmission. Copyright © 2011 Elsevier B.V. All rights reserved.

In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H+ exchanger (Pfnhe-1) gene: the absence of association in clinical isolates from the Republic of Congo

PubMed Central

2011-01-01

Background Quinine is still recommended as an effective therapy for severe cases of Plasmodium falciparum malaria, but the parasite has developed resistance to the drug in some cases. Investigations into the genetic basis for quinine resistance (QNR) suggest that QNR is complex and involves several genes, with either an additive or a pairwise effect. The results obtained when assessing one of these genes, the plasmodial Na+/H+ exchanger, Pfnhe-1, were found to depend upon the geographic origin of the parasite strain. Most of the associations identified have been made in Asian strains; in contrast, in African strains, the influence of Pfnhe on QNR is not apparent. However, a recent study carried out in Kenya did show a significant association between a Pfnhe polymorphism and QNR. As genetic differences may exist across the African continent, more field data are needed to determine if this association exists in other African regions. In the present study, association between Pfnhe and QNR is investigated in a series of isolates from central Africa. Methods The sequence analysis of the polymorphisms at the Pfnhe-1 ms4760 microsatellite and the evaluation of in vitro quinine susceptibility (by isotopic assay) were conducted in 74 P. falciparum isolates from the Republic of Congo. Results Polymorphisms in the number of DNNND or NHNDNHNNDDD repeats in the Pfnhe-1 ms4760 microsatellite were not associated with quinine susceptibility. Conclusions The polymorphism in the microsatellite ms4760 in Pfnhe-1 that cannot be used to monitor quinine response in the regions of the Republic of Congo, where the isolates came from. This finding suggests that there exists a genetic background associated with geographic area for the association that will prevent the use of Pfnhe as a molecular marker for QNR. The contribution of Pfnhe to the in vitro response to quinine remains to be assessed in other regions, including in countries with different levels of drug pressure. PMID:21314947

Chloroquine-resistant falciparum malaria in East Kalimantan, Indonesia.

PubMed

Verdrager, J; Arwati; Simanjuntak, C H; Saroso, J S

1976-03-01

Following the discovery of four imported chloroquine-resistant P. falciparum infections in the Province of Yogyakarta (Island of Java) sensitivity tests were carried out in the Province of East Kalimantan Island of Borneo). Twenty subjects were given 25 mg. of chloroquine base per kilogram of body weight over three days. Two infections were found resistant at the RII level and a third at the RI level with early recrudescence on day 7. In the other 17 cases followed up to day 21, six were found again with asexual parasites between day 9 and day 14 and a seventh on day 21. These results confirm the presence of chloroquine resistance in P. falciparum in East Kalimantan and, together with previous findings, suggest a widespread distribution of chloroquine-resistant falciparum malaria in this Province of Indonesia. It is particularly interesting to note that chloroquine-resistant falciparum malaria has now been detected in almost all the area of dispersion of A. balabacensis.

Plasmodium falciparum Malaria Endemicity in Indonesia in 2010

PubMed Central

Elyazar, Iqbal R. F.; Gething, Peter W.; Patil, Anand P.; Rogayah, Hanifah; Kusriastuti, Rita; Wismarini, Desak M.; Tarmizi, Siti N.; Baird, J. Kevin; Hay, Simon I.

2011-01-01

Background Malaria control programs require a detailed understanding of the contemporary spatial distribution of infection risk to efficiently allocate resources. We used model based geostatistics (MBG) techniques to generate a contemporary map of Plasmodium falciparum malaria risk in Indonesia in 2010. Methods Plasmodium falciparum Annual Parasite Incidence (PfAPI) data (2006–2008) were used to map limits of P. falciparum transmission. A total of 2,581 community blood surveys of P. falciparum parasite rate (PfPR) were identified (1985–2009). After quality control, 2,516 were included into a national database of age-standardized 2–10 year old PfPR data (PfPR2–10) for endemicity mapping. A Bayesian MBG procedure was used to create a predicted surface of PfPR2–10 endemicity with uncertainty estimates. Population at risk estimates were derived with reference to a 2010 human population count surface. Results We estimate 132.8 million people in Indonesia, lived at risk of P. falciparum transmission in 2010. Of these, 70.3% inhabited areas of unstable transmission and 29.7% in stable transmission. Among those exposed to stable risk, the vast majority were at low risk (93.39%) with the reminder at intermediate (6.6%) and high risk (0.01%). More people in western Indonesia lived in unstable rather than stable transmission zones. In contrast, fewer people in eastern Indonesia lived in unstable versus stable transmission areas. Conclusion While further feasibility assessments will be required, the immediate prospects for sustained control are good across much of the archipelago and medium term plans to transition to the pre-elimination phase are not unrealistic for P. falciparum. Endemicity in areas of Papua will clearly present the greatest challenge. This P. falciparum endemicity map allows malaria control agencies and their partners to comprehensively assess the region-specific prospects for reaching pre-elimination, monitor and evaluate the effectiveness of

Structure-activity analysis of CJ-15,801 analogues that interact with Plasmodium falciparum pantothenate kinase and inhibit parasite proliferation.

PubMed

Spry, Christina; Sewell, Alan L; Hering, Yuliya; Villa, Mathew V J; Weber, Jonas; Hobson, Stephen J; Harnor, Suzannah J; Gul, Sheraz; Marquez, Rodolfo; Saliba, Kevin J

2018-01-01

Survival of the human malaria parasite Plasmodium falciparum is dependent on pantothenate (vitamin B 5 ), a precursor of the fundamental enzyme cofactor coenzyme A. CJ-15,801, an enamide analogue of pantothenate isolated from the fungus Seimatosporium sp. CL28611, was previously shown to inhibit P. falciparum proliferation in vitro by targeting pantothenate utilization. To inform the design of next generation analogues, we set out to synthesize and test a series of synthetic enamide-bearing pantothenate analogues. We demonstrate that conservation of the R-pantoyl moiety and the trans-substituted double bond of CJ-15,801 is important for the selective, on-target antiplasmodial effect, while replacement of the carboxyl group is permitted, and, in one case, favored. Additionally, we show that the antiplasmodial potency of CJ-15,801 analogues that retain the R-pantoyl and trans-substituted enamide moieties correlates with inhibition of P. falciparum pantothenate kinase (PfPanK)-catalyzed pantothenate phosphorylation, implicating the interaction with PfPanK as a key determinant of antiplasmodial activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Specific receptors for phorbol diesters on freshly isolated human myeloid and lymphoid leukemia cells: comparable binding characteristics despite different cellular responses.

PubMed

Goodwin, B J; Moore, J O; Weinberg, J B

1984-02-01

Freshly isolated human leukemia cells have been shown in the past to display varying in vitro responses to phorbol diesters, depending on their cell type. Specific receptors for the phorbol diesters have been demonstrated on numerous different cells. This study was designed to characterize the receptors for phorbol diesters on leukemia cells freshly isolated from patients with different kinds of leukemia and to determine if differences in binding characteristics for tritium-labeled phorbol 12,13-dibutyrate (3H-PDBu) accounted for the different cellular responses elicited in vitro by phorbol diesters. Cells from 26 patients with different kinds of leukemia were studied. PDBu or phorbol 12-myristate 13-acetate (PMA) caused cells from patients with acute myeloblastic leukemia (AML), acute promyelocytic (APML), acute myelomonocytic (AMML), acute monocytic (AMoL), acute erythroleukemia (AEL), chronic myelocytic leukemia (CML) in blast crisis (myeloid), acute undifferentiated leukemia (AUL), and hairy cell leukemia (HCL) (n = 15) to adhere to plastic and spread. However, they caused no adherence or spreading and only slight aggregation of cells from patients with acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or CML-blast crisis (lymphoid) (n = 11). All leukemia cells studied, irrespective of cellular type, displayed specific receptors for 3H-PDBu. The time courses for binding by all leukemia types were similar, with peak binding at 5-10 min at 37 degrees C and 120 min at 4 degrees C. The binding affinities were similar for patients with ALL (96 +/- 32 nM, n = 4), CLL (126 +/- 32 nM, n = 6), and acute nonlymphoid leukemia (73 +/- 14 nM, n = 11). Likewise, the numbers of specific binding sites/cell were comparable for the patients with ALL (6.2 +/- 1.3 X 10(5) sites/cell, n = 4), CLL (5.0 +/- 2.0 X 10(5) sites/cell, n = 6), and acute nonlymphoid leukemia (4.4 +/- 1.9 X 10(5) sites/cell, n = 11). Thus, the differing responses to phorbol diesters of

Partner-Drug Resistance and Population Substructuring of Artemisinin-Resistant Plasmodium falciparum in Cambodia

PubMed Central

Parobek, Christian M.; Parr, Jonathan B.; Brazeau, Nicholas F.; Lon, Chanthap; Chaorattanakawee, Suwanna; Gosi, Panita; Barnett, Eric J.; Norris, Lauren D.; Meshnick, Steven R.; Spring, Michele D.; Lanteri, Charlotte A.; Bailey, Jeffrey A.; Saunders, David L.; Lin, Jessica T.

2017-01-01

Abstract Plasmodium falciparum in western Cambodia has developed resistance to artemisinin and its partner drugs, causing frequent treatment failure. Understanding this evolution can inform the deployment of new therapies. We investigated the genetic architecture of 78 falciparum isolates using whole-genome sequencing, correlating results to in vivo and ex vivo drug resistance and exploring the relationship between population structure, demographic history, and partner drug resistance. Principle component analysis, network analysis and demographic inference identified a diverse central population with three clusters of clonally expanding parasite populations, each associated with specific K13 artemisinin resistance alleles and partner drug resistance profiles which were consistent with the sequential deployment of artemisinin combination therapies in the region. One cluster displayed ex vivo piperaquine resistance and mefloquine sensitivity with a high rate of in vivo failure of dihydroartemisinin-piperaquine. Another cluster displayed ex vivo mefloquine resistance and piperaquine sensitivity with high in vivo efficacy of dihydroartemisinin-piperaquine. The final cluster was clonal and displayed intermediate sensitivity to both drugs. Variations in recently described piperaquine resistance markers did not explain the difference in mean IC90 or clinical failures between the high and intermediate piperaquine resistance groups, suggesting additional loci may be involved in resistance. The results highlight an important role for partner drug resistance in shaping the P. falciparum genetic landscape in Southeast Asia and suggest that further work is needed to evaluate for other mutations that drive piperaquine resistance. PMID:28854635

Computational synchronization of microarray data with application to Plasmodium falciparum.

PubMed

Zhao, Wei; Dauwels, Justin; Niles, Jacquin C; Cao, Jianshu

2012-06-21

Microarrays are widely used to investigate the blood stage of Plasmodium falciparum infection. Starting with synchronized cells, gene expression levels are continually measured over the 48-hour intra-erythrocytic cycle (IDC). However, the cell population gradually loses synchrony during the experiment. As a result, the microarray measurements are blurred. In this paper, we propose a generalized deconvolution approach to reconstruct the intrinsic expression pattern, and apply it to P. falciparum IDC microarray data. We develop a statistical model for the decay of synchrony among cells, and reconstruct the expression pattern through statistical inference. The proposed method can handle microarray measurements with noise and missing data. The original gene expression patterns become more apparent in the reconstructed profiles, making it easier to analyze and interpret the data. We hypothesize that reconstructed gene expression patterns represent better temporally resolved expression profiles that can be probabilistically modeled to match changes in expression level to IDC transitions. In particular, we identify transcriptionally regulated protein kinases putatively involved in regulating the P. falciparum IDC. By analyzing publicly available microarray data sets for the P. falciparum IDC, protein kinases are ranked in terms of their likelihood to be involved in regulating transitions between the ring, trophozoite and schizont developmental stages of the P. falciparum IDC. In our theoretical framework, a few protein kinases have high probability rankings, and could potentially be involved in regulating these developmental transitions. This study proposes a new methodology for extracting intrinsic expression patterns from microarray data. By applying this method to P. falciparum microarray data, several protein kinases are predicted to play a significant role in the P. falciparum IDC. Earlier experiments have indeed confirmed that several of these kinases are involved

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 fresh.â 101.95 Section 101.95 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... Descriptive Claims That Are Neither Nutrient Content Claims nor Health Claims § 101.95 “Fresh,” “freshly...

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 fresh.â 101.95 Section 101.95 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... Descriptive Claims That Are Neither Nutrient Content Claims nor Health Claims § 101.95 “Fresh,” “freshly...

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 fresh.â 101.95 Section 101.95 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... Descriptive Claims That Are Neither Nutrient Content Claims nor Health Claims § 101.95 “Fresh,” “freshly...

On Programmed Cell Death in Plasmodium falciparum: Status Quo

PubMed Central

Engelbrecht, Dewaldt; Durand, Pierre Marcel; Coetzer, Thérèsa Louise

2012-01-01

Conflicting arguments and results exist regarding the occurrence and phenotype of programmed cell death (PCD) in the malaria parasite Plasmodium falciparum. Inconsistencies relate mainly to the number and type of PCD markers assessed and the different methodologies used in the studies. In this paper, we provide a comprehensive overview of the current state of knowledge and empirical evidence for PCD in the intraerythrocytic stages of P. falciparum. We consider possible reasons for discrepancies in the data and offer suggestions towards more standardised investigation methods in this field. Furthermore, we present genomic evidence for PCD machinery in P. falciparum. We discuss the potential adaptive or nonadaptive role of PCD in the parasite life cycle and its possible exploitation in the development of novel drug targets. Lastly, we pose pertinent unanswered questions concerning the PCD phenomenon in P. falciparum to provide future direction. PMID:22287973

Enhancing longevity of Plasmodium vivax and P. falciparum sporozoites after dissection from mosquito salivary glands.

PubMed

Lupton, Emily J; Roth, Alison; Patrapuvich, Rapatbhorn; Maher, Steve P; Singh, Naresh; Sattabongkot, Jetsumon; Adams, John H

2015-04-01

The pre-erythrocytic stages of Plasmodium vivax and Plasmodium falciparum remain challenging for experimental research in part due to limited access to sporozoites. An important factor limiting availability is the laboratory support required for producing infected mosquitoes and the ephemeral nature of isolated extracellular sporozoites. This study was undertaken to investigate methods to improve the availability of this limited resource by extending the longevity of the extracellular sporozoites after mosquito dissection. Our goal in this study was to determine whether buffer conditions more closely mimicking the insect microenvironment could prolong longevity of ex vivo P. vivax and P. falciparum sporozoites. The study compared the current standard dissection buffer RPMI1640 to Hank's Balanced Salt Solution with 1g/L glucose (HBSS-1) or 2g/L glucose (HBSS-2) and Grace's Insect Medium for ability to extend longevity of ex vivo P. vivax and P. falciparum sporozoites. The effect of each buffer on sporozoite viability was evaluated by measuring sporozoite gliding motility at 0, 4, 8, and 24h post-dissection from mosquito salivary glands. Comparisons of mean gliding percentages of ex vivo sporozoites in the different buffers and time points found that RPMI and Grace's both showed strong gliding at 0h. In contrast, by 4h post-dissection sporozoites in RPMI consistently had the lowest gliding activity, whereas sporozoites in Grace's had significantly more gliding compared to all other buffers at almost all time points. Our results indicate that P. vivax and P. falciparum sporozoites maintained in insect media rather than the standard dissection buffer RPMI and HBSS retain viability better over time. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

Pathogenicity Determinants of the Human Malaria Parasite Plasmodium falciparum Have Ancient Origins

PubMed Central

Brazier, Andrew J.; Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell

2017-01-01

ABSTRACT Plasmodium falciparum, the most deadly of the human malaria parasites, is a member of the Laverania subgenus that also infects African Great Apes. The virulence of P. falciparum is related to cytoadhesion of infected erythrocytes in microvasculature, but the origin of dangerous parasite adhesion traits is poorly understood. To investigate the evolutionary history of the P. falciparum cytoadhesion pathogenicity determinant, we studied adhesion domains from the chimpanzee malaria parasite P. reichenowi. We demonstrate that the P. reichenowi var gene repertoire encodes cysteine-rich interdomain region (CIDR) domains which bind human CD36 and endothelial protein C receptor (EPCR) with the same levels of affinity and at binding sites similar to those bound by P. falciparum. Moreover, P. reichenowi domains interfere with the protective function of the activated protein C-EPCR pathway on endothelial cells, a presumptive virulence trait in humans. These findings provide evidence for ancient evolutionary origins of two key cytoadhesion properties of P. falciparum that contribute to human infection and pathogenicity. IMPORTANCE Cytoadhesion of P. falciparum-infected erythrocytes in the microcirculation is a major virulence determinant. P. falciparum is descended from a subgenus of parasites that also infect chimpanzees and gorillas and exhibits strict host species specificity. Despite their high genetic similarity to P. falciparum, it is unknown whether ape parasites encode adhesion properties similar to those of P. falciparum or are as virulent in their natural hosts. Consequently, it has been unclear when virulent adhesion traits arose in P. falciparum and how long they have been present in the parasite population. It is also unknown whether cytoadhesive interactions pose a barrier to cross-species transmission. We show that parasite domains from the chimpanzee malaria parasite P. reichenowi bind human receptors with specificity similar to that of P. falciparum

Pathogenicity Determinants of the Human Malaria Parasite Plasmodium falciparum Have Ancient Origins.

PubMed

Brazier, Andrew J; Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell; Smith, Joseph D

2017-01-01

Plasmodium falciparum , the most deadly of the human malaria parasites, is a member of the Laverania subgenus that also infects African Great Apes. The virulence of P. falciparum is related to cytoadhesion of infected erythrocytes in microvasculature, but the origin of dangerous parasite adhesion traits is poorly understood. To investigate the evolutionary history of the P. falciparum cytoadhesion pathogenicity determinant, we studied adhesion domains from the chimpanzee malaria parasite P. reichenowi . We demonstrate that the P. reichenowi var gene repertoire encodes cysteine-rich interdomain region (CIDR) domains which bind human CD36 and endothelial protein C receptor (EPCR) with the same levels of affinity and at binding sites similar to those bound by P. falciparum . Moreover, P. reichenowi domains interfere with the protective function of the activated protein C-EPCR pathway on endothelial cells, a presumptive virulence trait in humans. These findings provide evidence for ancient evolutionary origins of two key cytoadhesion properties of P. falciparum that contribute to human infection and pathogenicity. IMPORTANCE Cytoadhesion of P. falciparum -infected erythrocytes in the microcirculation is a major virulence determinant. P. falciparum is descended from a subgenus of parasites that also infect chimpanzees and gorillas and exhibits strict host species specificity. Despite their high genetic similarity to P. falciparum , it is unknown whether ape parasites encode adhesion properties similar to those of P. falciparum or are as virulent in their natural hosts. Consequently, it has been unclear when virulent adhesion traits arose in P. falciparum and how long they have been present in the parasite population. It is also unknown whether cytoadhesive interactions pose a barrier to cross-species transmission. We show that parasite domains from the chimpanzee malaria parasite P. reichenowi bind human receptors with specificity similar to that of P. falciparum

Drug Evaluation in the Plasmodium falciparum - Aotus Model

DTIC Science & Technology

1984-09-01

consecutive days to Colombian Aotus. Six amodiaquin analogues were evaluated for their capacity to cure in- fections of chloroquine -sensitive and...AMODIAQUIN ANALOGUES AND AMODIAQUIN AGAINST INFECTIONS OF CHLOROQUINE -SENSITIVE AND CHLOROQUINE -RESISTANT STRAINS OF PLASMODIUM FALCIPARUM 14...AMODIAQUIN ANALOGUES AND AMOOIAQUIN AGAINST INFECTIONS OF CHLOROQUINE -SENSITIVE AND CHLOROQUINE - RESISTANT STRAINS OF PLASMODIUM FALCIPARUM Following

Characterization of antimicrobial resistance and quinolone resistance factors in high-level ciprofloxacin-resistant Enterococcus faecalis and Enterococcus faecium isolates obtained from fresh produce and fecal samples of patients.

PubMed

Kim, Min-Chan; Woo, Gun-Jo

2017-07-01

The emergence of fluoroquinolone-resistant enterococci is worldwide. Antimicrobial resistance was characterized and the effect of quinolone-resistance factors was analyzed in high-level ciprofloxacin-resistant (HLCR) Enterococcus faecalis and Enterococcus faecium isolated from fresh produce and fecal samples of patients. Among the 81 ciprofloxacin-resistant Enterococcus isolates, 46 showed high levels of ciprofloxacin resistance, resistance to other quinolone antibiotics, and multidrug resistance profiles. The virulence factors esp and hyl were identified in 27 (58.7%) and 25 (54.3%) of isolates, respectively. Sequence type analysis showed that 35 strains of HLCR E. faecium were clonal complex 17. Eleven strains of HLCR E. faecalis were confirmed as sequence type (ST) 28, ST 64 and ST 125. Quinolone resistance-determining region mutation was identified in HLCR Enterococcus isolates; with serine being changed in gyrA83, gyrA87 and parC80. This result shows that gyrA and parC mutations could be important factors for high-level resistance to fluoroquinolones. No significant differences were observed in antimicrobial resistance patterns and genetic characteristics among the isolates from fresh produce and fecal samples. Therefore, good agricultural practices in farming and continuous monitoring of patients, food and the environment for Enterococcus spp. should be performed to prevent antimicrobial resistance and enable reduction of resistance rates. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum*

PubMed Central

Cassera, María B.; Hazleton, Keith Z.; Riegelhaupt, Paul M.; Merino, Emilio F.; Luo, Minkui; Akabas, Myles H.; Schramm, Vern L.

2008-01-01

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum. PMID:18799466

Erythrocytic adenosine monophosphate as an alternative purine source in Plasmodium falciparum.

PubMed

Cassera, María B; Hazleton, Keith Z; Riegelhaupt, Paul M; Merino, Emilio F; Luo, Minkui; Akabas, Myles H; Schramm, Vern L

2008-11-21

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

PubMed

Wimmer, Isabella; Tröscher, Anna R; Brunner, Florian; Rubino, Stephen J; Bien, Christian G; Weiner, Howard L; Lassmann, Hans; Bauer, Jan

2018-04-20

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

PubMed

Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

2016-11-01

Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

Identification of resistance of Plasmodium falciparum to artesunate-mefloquine combination in an area along the Thai-Myanmar border: integration of clinico-parasitological response, systemic drug exposure, and in vitro parasite sensitivity

PubMed Central

2013-01-01

Background A markedly high failure rate of three-day artesunate-mefloquine was observed in the area along the Thai-Myanmar border. Methods Identification of Plasmodium falciparum isolates with intrinsic resistance to each component of the artesunate-mefloquine combination was analysed with integrated information on clinico-parasitological response, together with systemic drug exposure (area under blood/plasma concentration-time curves (AUC)) of dihydroartemisinin and mefloquine, and in vitro sensitivity of P. falciparum in a total of 17 out of 29 P. falciparum isolates from patients with acute uncomplicated falciparum malaria. Analysis of the contribution of in vitro parasite sensitivity and systemic drug exposure and relationship with pfmdr1 copy number in the group with sensitive response was performed in 21 of 69 cases. Results Identification of resistance and/or reduced intrinsic parasitocidal activity of artesunate and/or mefloquine without pharmacokinetic or other host-related factors were confirmed in six cases: one with reduced sensitivity to artesunate alone, two with resistance to mefloquine alone, and three with reduced sensitivity to artesunate combined with resistance to mefloquine. Resistance and/or reduced intrinsic parasitocidal activity of mefloquine/artesunate, together with contribution of pharmacokinetic factor of mefloquine and/or artesunate were identified in seven cases: two with resistance to mefloquine alone, and five with resistance to mefloquine combined with reduced sensitivity to artesunate. Pharmacokinetic factor alone contributed to recrudescence in three cases, all of which had inadequate whole blood mefloquine levels (AUC0-7days). Other host-related factors contributed to recrudescence in one case. Amplification of pfmdr1 (increasing of pfmdr1 copy number) is a related molecular marker of artesunate-mefloquine resistance and seems to be a suitable molecular marker to predict occurrence of recrudescence. Conclusions Despite the

Real-time polymerase chain reaction assay for the rapid detection and characterization of chloroquine-resistant Plasmodium falciparum malaria in returned travelers.

PubMed

Farcas, Gabriella A; Soeller, Rainer; Zhong, Kathleen; Zahirieh, Alireza; Kain, Kevin C

2006-03-01

Imported drug-resistant malaria is a growing problem in industrialized countries. Rapid and accurate diagnosis is essential to prevent malaria-associated mortality in returned travelers. However, outside of a limited number of specialized centers, the microscopic diagnosis of malaria is slow, unreliable, and provides little information about drug resistance. Molecular diagnostics have the potential to overcome these limitations. We developed and evaluated a rapid, real-time polymerase chain reaction (PCR) assay to detect Plasmodium falciparum malaria and chloroquine (CQ)-resistance determinants in returned travelers who are febrile. A real-time PCR assay based on detection of the K76T mutation in PfCRT (K76T) of P. falciparum was developed on a LightCycler platform (Roche). The performance characteristics of the real-time assay were compared with those of the nested PCR-restriction fragment-length polymorphism (RFLP) and the sequence analyses of samples obtained from 200 febrile returned travelers, who included 125 infected with P. falciparum (48 of whom were infected CQ-susceptible [K76] and 77 of whom were CQ-resistant [T76] P. falciparum), 22 infected with Plasmodium vivax, 10 infected with Plasmodium ovale, 3 infected with Plasmodium malariae malaria, and 40 infected with other febrile syndromes. All patient samples were coded, and all analyses were performed blindly. The real-time PCR assay detected multiple pfcrt haplotypes associated with CQ resistance in geographically diverse malaria isolates acquired by travelers. Compared with nested-PCR RFLP (the reference standard), the real-time assay was 100% sensitive and 96.2% specific for detection of the P. falciparum K76T mutation. This assay is rapid, sensitive, and specific for the detection and characterization of CQ-resistant P. falciparum malaria in returned travelers. This assay is automated, standardized, and suitable for routine use in clinical diagnostic laboratories.

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. Copyright �

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

Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification.

PubMed

Poon, Leo L M; Wong, Bonnie W Y; Ma, Edmund H T; Chan, Kwok H; Chow, Larry M C; Abeyewickreme, Wimal; Tangpukdee, Noppadon; Yuen, Kwok Y; Guan, Yi; Looareesuwan, Sornchai; Peiris, J S Malik

2006-02-01

Malaria is one of the most important parasitic infections in humans. A sensitive diagnostic test for malaria that could be applied at the community level could be useful in programs to control the disease. The aim of the present work was to develop a simple, inexpensive molecular test for Plasmodium falciparum. Blood was collected from controls (n = 100) and from patients diagnosed with falciparum malaria infection (n = 102), who were recruited to the study. Heat-treated blood samples were tested by a loop-mediated isothermal amplification (LAMP) assay for P. falciparum. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. To evaluate the assay, DNA from these samples was purified and tested by PCR. Results from the LAMP and PCR assays were compared. The LAMP assay detected P. falciparum directly from heat-treated blood. The quantitative data from the assay correlated to the parasite counts obtained by blood-film microscopic analyses. When we used the PCR assay as the comparison method, the sensitivity and specificity of the LAMP assay were 95% and 99%, respectively. Unlike PCR, the LAMP assay does not require purified DNA for efficient DNA amplification, thereby reducing the cost and turnaround time for P. falciparum diagnosis. The assay requires only basic instruments, and assay positivity can be verified by visual inspection.

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels.

PubMed

Brzica, Hrvoje; Abdullahi, Wazir; Reilly, Bianca G; Ronaldson, Patrick T

2018-05-07

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

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

Microsatellite Genotyping of Plasmodium vivax Isolates from Pregnant Women in Four Malaria Endemic Countries

PubMed Central

Menegon, Michela; Bardají, Azucena; Martínez-Espinosa, Flor; Bôtto-Menezes, Camila; Ome-Kaius, Maria; Mueller, Ivo; Betuela, Inoni; Arévalo-Herrera, Myriam; Kochar, Swati; Kochar, Sanjay K.; Jaju, Puneet; Hans, Dhiraj; Chitnis, Chetan; Padilla, Norma; Castellanos, María Eugenia; Ortiz, Lucía; Sanz, Sergi; Piqueras, Mireia; Desai, Meghna; Mayor, Alfredo; del Portillo, Hernando; Menéndez, Clara; Severini, Carlo

2016-01-01

Plasmodium vivax is the most widely distributed human parasite and the main cause of human malaria outside the African continent. However, the knowledge about the genetic variability of P. vivax is limited when compared to the information available for P. falciparum. We present the results of a study aimed at characterizing the genetic structure of P. vivax populations obtained from pregnant women from different malaria endemic settings. Between June 2008 and October 2011 nearly 2000 pregnant women were recruited during routine antenatal care at each site and followed up until delivery. A capillary blood sample from the study participants was collected for genotyping at different time points. Seven P. vivax microsatellite markers were used for genotypic characterization on a total of 229 P. vivax isolates obtained from Brazil, Colombia, India and Papua New Guinea. In each population, the number of alleles per locus, the expected heterozygosity and the levels of multilocus linkage disequilibrium were assessed. The extent of genetic differentiation among populations was also estimated. Six microsatellite loci on 137 P. falciparum isolates from three countries were screened for comparison. The mean value of expected heterozygosity per country ranged from 0.839 to 0.874 for P. vivax and from 0.578 to 0.758 for P. falciparum. P. vivax populations were more diverse than those of P. falciparum. In some of the studied countries, the diversity of P. vivax population was very high compared to the respective level of endemicity. The level of inter-population differentiation was moderate to high in all P. vivax and P. falciparum populations studied. PMID:27011010

Drug Evaluation in the Plasmodium Falciparum - Aotus Model

DTIC Science & Technology

1994-03-15

falciparum infections. Althogh erythromycin is inactive against chloroquine -resistant falciparum infections, an analogue , azithromycin, is effective in vitro...response to chloroquine , and then expand the evaluation of WR 238605, a primaquine analogue against infections. Each cyopreserved sample was thawed rapidly...confirmedo.4 chloroquine -sensitive p. via -strai-n[as not Infective for unaltered Panamanian Aotus. 14. SUBJECT TERMS 15. NUMBER OF PAGES Malaria

Elimination of Plasmodium falciparum malaria in Tajikistan.

PubMed

Kondrashin, Anatoly V; Sharipov, Azizullo S; Kadamov, Dilshod S; Karimov, Saifuddin S; Gasimov, Elkhan; Baranova, Alla M; Morozova, Lola F; Stepanova, Ekaterina V; Turbabina, Natalia A; Maksimova, Maria S; Morozov, Evgeny N

2017-05-30

Malaria was eliminated in Tajikistan by the beginning of the 1960s. However, sporadic introduced cases of malaria occurred subsequently probably as a result of transmission from infected mosquito Anopheles flying over river the Punj from the border areas of Afghanistan. During the 1970s and 1980s local outbreaks of malaria were reported in the southern districts bordering Afghanistan. The malaria situation dramatically changed during the 1990s following armed conflict and civil unrest in the newly independent Tajikistan, which paralyzed health services including the malaria control activities and a large-scale malaria epidemic occurred with more than 400,000 malaria cases. The malaria epidemic was contained by 1999 as a result of considerable financial input from the Government and the international community. Although Plasmodium falciparum constituted only about 5% of total malaria cases, reduction of its incidence was slower than that of Plasmodium vivax. To prevent increase in P. falciparum malaria both in terms of incidence and territory, a P. falciparum elimination programme in the Republic was launched in 200, jointly supported by the Government and the Global Fund for control of AIDS, tuberculosis and malaria. The main activities included the use of pyrethroids for the IRS with determined periodicity, deployment of mosquito nets, impregnated with insecticides, use of larvivorous fishes as a biological larvicide, implementation of small-scale environmental management, and use of personal protection methods by population under malaria risk. The malaria surveillance system was strengthened by the use of ACD, PCD, RCD and selective use of mass blood surveys. All detected cases were timely epidemiologically investigated and treated based on the results of laboratory diagnosis. As a result, by 2009, P. falciparum malaria was eliminated from all of Tajikistan, one year ahead of the originally targeted date. Elimination of P. falciparum also contributed towards

In vitro antiplasmodial activity of bacterium RJAUTHB 14 associated with marine sponge Haliclona Grant against Plasmodium falciparum.

PubMed

Jacob Inbaneson, Samuel; Ravikumar, Sundaram

2012-06-01

Malaria is the most important parasitic disease, leading to annual death of about one million people, and the Plasmodium falciparum develops resistance to well-established antimalarial drugs. The newest antiplasmodial drug from a marine microorganism helps in addressing this problem. In the present study, Haliclona Grant were collected and subjected for enumeration and isolation of associated bacteria. The count of bacterial isolates was maximum in November 2007 (18 × 10(4) colony-forming units (CFU) g(-1), and the average count was maximum during the monsoon season (117 × 10(3) CFU g(-1)). Thirty-three morphologically different bacterial isolates were isolated from Haliclona Grant, and the extracellular ethyl acetate extracts were screened for antiplasmodial activity against P. falciparum. The antiplasmodial activity of bacterium RJAUTHB 14 (11.98 μg[Symbol: see text]ml(-1)) is highly comparable with the positive control chloroquine (IC(50) 19.59 μg[Symbol: see text]ml(-1)), but the other 21 bacterial extracts showed an IC(50) value of more than 100 μg[Symbol: see text]ml(-1). Statistical analysis reveals that significant in vitro antiplasmodial activity (P < 0.05) was observed between the concentrations and time of exposure. The chemical injury to erythrocytes showed no morphological changes in erythrocytes by the ethyl acetate extract of bacterial isolates after 48 h of incubation. The in vitro antiplasmodial activity might be due to the presence of reducing sugars and alkaloids in the ethyl acetate extracts of bacterium RJAUTHB 14. The 16S rRNA gene partial sequence of bacterium RJAUTHB 14 is deposited in NCBI (GenBank accession no. GU269569). It is concluded from the present study that the ethyl acetate extracts of bacterium RJAUTHB 14 possess lead compounds for the development of antiplasmodial drugs.

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

An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer.

PubMed

Imai, Kazuo; Tarumoto, Norihito; Runtuwene, Lucky Ronald; Sakai, Jun; Hayashida, Kyoko; Eshita, Yuki; Maeda, Ryuichiro; Tuda, Josef; Ohno, Hideaki; Murakami, Takashi; Maesaki, Shigefumi; Suzuki, Yutaka; Yamagishi, Junya; Maeda, Takuya

2018-05-29

The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.

Development of cultured Plasmodium falciparum blood-stage malaria cell banks for early phase in vivo clinical trial assessment of anti-malaria drugs and vaccines.

PubMed

Stanisic, Danielle I; Liu, Xue Q; De, Sai Lata; Batzloff, Michael R; Forbes, Tanya; Davis, Christopher B; Sekuloski, Silvana; Chavchich, Marina; Chung, Wendy; Trenholme, Katharine; McCarthy, James S; Li, Tao; Sim, B Kim Lee; Hoffman, Stephen L; Good, Michael F

2015-04-07

The ability to undertake controlled human malaria infection (CHMI) studies for preliminary evaluation of malaria vaccine candidates and anti-malaria drug efficacy has been limited by the need for access to sporozoite infected mosquitoes, aseptic, purified, cryopreserved sporozoites or blood-stage malaria parasites derived ex vivo from malaria infected individuals. Three different strategies are described for the manufacture of clinical grade cultured malaria cell banks suitable for use in CHMI studies. Good Manufacturing Practices (GMP)-grade Plasmodium falciparum NF54, clinically isolated 3D7, and research-grade P. falciparum 7G8 blood-stage malaria parasites were cultured separately in GMP-compliant facilities using screened blood components and then cryopreserved to produce three P. falciparum blood-stage malaria cell banks. These cell banks were evaluated according to specific criteria (parasitaemia, identity, viability, sterility, presence of endotoxin, presence of mycoplasma or other viral agents and in vitro anti-malarial drug sensitivity of the cell bank malaria parasites) to ensure they met the criteria to permit product release according to GMP requirements. The P. falciparum NF54, 3D7 and 7G8 cell banks consisted of >78% ring stage parasites with a ring stage parasitaemia of >1.4%. Parasites were viable in vitro following thawing. The cell banks were free from contamination with bacteria, mycoplasma and a broad panel of viruses. The P. falciparum NF54, 3D7 and 7G8 parasites exhibited differential anti-malarial drug susceptibilities. The P. falciparum NF54 and 3D7 parasites were susceptible to all anti-malaria compounds tested, whereas the P. falciparum 7G8 parasites were resistant/had decreased susceptibility to four compounds. Following testing, all defined release criteria were met and the P. falciparum cell banks were deemed suitable for release. Ethical approval has been obtained for administration to human volunteers. The production of cultured P

Genetic diversity of Plasmodium falciparum merozoite surface protein-1 (block 2), glutamate-rich protein and sexual stage antigen Pfs25 from Chandigarh, North India.

PubMed

Kaur, Hargobinder; Sehgal, Rakesh; Goyal, Kapil; Makkar, Nikita; Yadav, Richa; Bharti, Praveen K; Singh, Neeru; Sarmah, Nilanju P; Mohapatra, Pradyumna K; Mahanta, Jagadish; Bansal, Devendra; Sultan, Ali A; Kanwar, Jagat R

2017-12-01

To elucidate the genetic diversity of Plasmodium falciparum in residual transmission foci of northern India. Clinically suspected patients with malaria were screened for malaria infection by microscopy. 48 P. falciparum-infected patients were enrolled from tertiary care hospital in Chandigarh, India. Blood samples were collected from enrolled patients, genomic DNA extraction and nested PCR was performed for further species confirmation. Sanger sequencing was carried out using block 2 region of msp1, R2 region of glurp and pfs25-specific primers. Extensive diversity was found in msp1 alleles with predominantly RO33 alleles. Overall allelic prevalence was 55.8% for RO33, 39.5% for MAD20 and 4.7% for K1. Six variants were observed in MAD20, whereas no variant was found in RO33 and K1 alleles. A phylogenetic analysis of RO33 alleles indicated more similarity to South African isolates, whereas MAD20 alleles showed similarity with South-East Asian isolates. In glurp, extensive variation was observed with eleven different alleles based on the AAU repeats. However, pfs25 showed less diversity and was the most stable among the targeted genes. Our findings document the genetic diversity among circulating strains of P. falciparum in an area of India with low malaria transmission and could have implications for control strategies to reach the national goal of malaria elimination. © 2017 John Wiley & Sons Ltd.

Novel Polymorphisms in Plasmodium falciparum ABC Transporter Genes Are Associated with Major ACT Antimalarial Drug Resistance

PubMed Central

Veiga, Maria Isabel; Ferreira, Pedro Eduardo; Jörnhagen, Louise; Malmberg, Maja; Kone, Aminatou; Schmidt, Berit Aydin; Petzold, Max; Björkman, Anders; Nosten, Francois; Gil, Jose Pedro

2011-01-01

Chemotherapy is a critical component of malaria control. However, the most deadly malaria pathogen, Plasmodium falciparum, has repeatedly mounted resistance against a series of antimalarial drugs used in the last decades. Southeast Asia is an epicenter of emerging antimalarial drug resistance, including recent resistance to the artemisinins, the core component of all recommended antimalarial combination therapies. Alterations in the parasitic membrane proteins Pgh-1, PfCRT and PfMRP1 are believed to be major contributors to resistance through decreasing intracellular drug accumulation. The pfcrt, pfmdr1 and pfmrp1 genes were sequenced from a set of P.falciparum field isolates from the Thai-Myanmar border. In vitro drug susceptibility to artemisinin, dihydroartemisinin, mefloquine and lumefantrine were assessed. Positive correlations were seen between the in vitro susceptibility responses to artemisinin and dihydroartemisinin and the responses to the arylamino-alcohol quinolines lumefantrine and mefloquine. The previously unstudied pfmdr1 F1226Y and pfmrp1 F1390I SNPs were associated significantly with artemisinin, mefloquine and lumefantrine in vitro susceptibility. A variation in pfmdr1 gene copy number was also associated with parasite drug susceptibility of artemisinin, mefloquine and lumefantrine. Our work unveils new candidate markers of P. falciparum multidrug resistance in vitro, while contributing to the understanding of subjacent genetic complexity, essential for future evidence-based drug policy decisions. PMID:21633513

Novel polymorphisms in Plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance.

PubMed

Veiga, Maria Isabel; Ferreira, Pedro Eduardo; Jörnhagen, Louise; Malmberg, Maja; Kone, Aminatou; Schmidt, Berit Aydin; Petzold, Max; Björkman, Anders; Nosten, Francois; Gil, Jose Pedro

2011-01-01

Chemotherapy is a critical component of malaria control. However, the most deadly malaria pathogen, Plasmodium falciparum, has repeatedly mounted resistance against a series of antimalarial drugs used in the last decades. Southeast Asia is an epicenter of emerging antimalarial drug resistance, including recent resistance to the artemisinins, the core component of all recommended antimalarial combination therapies. Alterations in the parasitic membrane proteins Pgh-1, PfCRT and PfMRP1 are believed to be major contributors to resistance through decreasing intracellular drug accumulation. The pfcrt, pfmdr1 and pfmrp1 genes were sequenced from a set of P.falciparum field isolates from the Thai-Myanmar border. In vitro drug susceptibility to artemisinin, dihydroartemisinin, mefloquine and lumefantrine were assessed. Positive correlations were seen between the in vitro susceptibility responses to artemisinin and dihydroartemisinin and the responses to the arylamino-alcohol quinolines lumefantrine and mefloquine. The previously unstudied pfmdr1 F1226Y and pfmrp1 F1390I SNPs were associated significantly with artemisinin, mefloquine and lumefantrine in vitro susceptibility. A variation in pfmdr1 gene copy number was also associated with parasite drug susceptibility of artemisinin, mefloquine and lumefantrine. Our work unveils new candidate markers of P. falciparum multidrug resistance in vitro, while contributing to the understanding of subjacent genetic complexity, essential for future evidence-based drug policy decisions.

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.

Steroidal saponins from fresh stems of Dracaena angustifolia

USDA-ARS?s Scientific Manuscript database

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...

Falciparum malaria infection with invasive pulmonary aspergillosis in immunocompetent host – case report

NASA Astrophysics Data System (ADS)

Andriyani, Y.

2018-03-01

Invasive pulmonary aspergillosis is an extraordinary rare in the immunocompetent host. Falciparum malaria contributes to high morbidity and mortality of malaria infection cases in the world. The impairments of both humoral and cellular immunity could be the reason of invasive pulmonary aspergillosis in falciparum malaria infection. Forty-nine years old patient came with fever, jaundice, pain in the right abdomen, after visiting a remote area in Africa about one month before admission. Blood films and rapid test were positive for Plasmodium falciparum. After malaria therapy in five days, consciousness was altered into somnolence and intubated with respiratory deterioration. Invasive pulmonary aspergillosis after falciparum malaria infection is life-threatening. There should be awareness of physicians of invasive pulmonary aspergillosis in falciparum malaria infection.

Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum

PubMed Central

Chiang, Annette N.; Valderramos, Juan-Carlos; Balachandran, Raghavan; Chovatiya, Raj J.; Mead, Brian P.; Schneider, Corinne; Bell, Samantha L.; Klein, Michael G.; Huryn, Donna M.; Chen, Xiaojiang S.; Day, Billy W.; Fidock, David A.; Wipf, Peter; Brodsky, Jeffrey L.

2009-01-01

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC50 values from 30 nM to 1.6 μM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents. PMID:19195901

NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children.

PubMed

Vignali, Marissa; Armour, Christopher D; Chen, Jingyang; Morrison, Robert; Castle, John C; Biery, Matthew C; Bouzek, Heather; Moon, Wonjong; Babak, Tomas; Fried, Michal; Raymond, Christopher K; Duffy, Patrick E

2011-03-01

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases.

NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children

PubMed Central

Vignali, Marissa; Armour, Christopher D.; Chen, Jingyang; Morrison, Robert; Castle, John C.; Biery, Matthew C.; Bouzek, Heather; Moon, Wonjong; Babak, Tomas; Fried, Michal; Raymond, Christopher K.; Duffy, Patrick E.

2011-01-01

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases. PMID:21317536

A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

PubMed Central

2012-01-01

Background Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated. Methods PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy. Results Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369), containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein. Conclusion Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds. PMID:23190769

Dibenzylideneacetone analogues as novel Plasmodium falciparum inhibitors.

PubMed

Aher, Rahul Balasaheb; Wanare, Gajanan; Kawathekar, Neha; Kumar, Ravi Ranjan; Kaushik, Naveen Kumar; Sahal, Dinkar; Chauhan, Virander Singh

2011-05-15

A series of dibenzylideneacetones (A1-A12) and some of their pyrazolines (B1-B4) were synthesized and evaluated in vitro for blood stage antiplasmodial properties in Plasmodium falciparum culture using SYBR-green-I fluorescence assay. The compound (1E, 4E)-1,5-bis(3,4-dimethoxyphenyl)penta-1,4-dien-3-one (A9) was found to be the most active with IC(50) of 1.97 μM against chloroquine-sensitive strain (3D7) and 1.69 μM against chloroquine-resistant field isolate (RKL9). The MTT based cytotoxicity assay on HeLa cell line has confirmed that A9 is selective in its action against malaria parasite (with a therapeutic index of 166). Our results revealed that these compounds exhibited promising antiplasmodial activities which can be further explored as potential leads for the development of cheaper, safe, effective and potent drugs against chloroquine-resistant malarial parasites. Copyright © 2011. Published by Elsevier Ltd.

High prevalence of asymptomatic Plasmodium falciparum infection in Gabonese adults.

PubMed

Dal-Bianco, Matthias P; Köster, Kai B; Kombila, Ulrich D; Kun, Jürgen F J; Grobusch, Martin P; Ngoma, Ghyslain Mombo; Matsiegui, Pierre B; Supan, Christian; Salazar, Carmen L Ospina; Missinou, Michel A; Issifou, Saadou; Lell, Bertrand; Kremsner, Peter

2007-11-01

Plasmodium falciparum, the most common malarial parasite in sub-Saharan Africa, accounts for a high number of deaths in children less than five years of age. In malaria-endemic countries with stable transmission, semi-immunity is usually acquired after childhood. For adults, severe malaria is rare. Infected adults have either uncomplicated malaria or asymptomatic parasitemia. During a period of one year, we screened 497 afebrile males to investigate the prevalence of asymptomatic P. falciparum parasitemia in villages near Lambaréné, Gabon by use of three different methods. A total of 52% of the individuals had parasites detected by a subtelomeric variable open reading frame polymerase chain reaction (stevor-PCR), 27% of the rapid diagnostic test results were positive, and 12% of the thick blood smears with low parasitemias had P. falciparum. Most positive cases were only detected by the stevor-PCR. Asymptomatic P. falciparum parasitemia in adults living in a malaria-endemic country is frequent.

Antibodies among men and children to placental-binding Plasmodium falciparum-infected erythrocytes that express var2csa.

PubMed

Beeson, James G; Ndungu, Francis; Persson, Kristina E M; Chesson, Joanne M; Kelly, Greg L; Uyoga, Sophie; Hallamore, Sandra L; Williams, Thomas N; Reeder, John C; Brown, Graham V; Marsh, Kevin

2007-07-01

During pregnancy, specific variants of Plasmodium falciparum-infected erythrocytes (IEs) can accumulate in the placenta through adhesion to chondroitin sulfate A (CSA) mediated by expression of PfEMP1 encoded by var2csa-type genes. Antibodies against these variants are associated with protection from maternal malaria. We evaluated antibodies among Kenyan, Papua New Guinean, and Malawian men and Kenyan children against two different CSA-binding P. falciparum isolates expressing var2csa variants. Specific IgG was present at significant levels among some men and children from each population, suggesting exposure to these variants is not exclusive to pregnancy. However, the level and prevalence of antibodies was substantially lower overall than exposed multigravidas. IgG-binding was specific and did not represent antibodies to subpopulations of non-CSA-binding IEs, and some sera inhibited IE adhesion to CSA. These findings have significant implications for understanding malaria pathogenesis and immunity and may be significant for understanding the acquisition of immunity to maternal malaria.

Plasmodium falciparum in the southeastern Atlantic forest: a challenge to the bromeliad-malaria paradigm?

PubMed

Laporta, Gabriel Zorello; Burattini, Marcelo Nascimento; Levy, Debora; Fukuya, Linah Akemi; de Oliveira, Tatiane Marques Porangaba; Maselli, Luciana Morganti Ferreira; Conn, Jan Evelyn; Massad, Eduardo; Bydlowski, Sergio Paulo; Sallum, Maria Anice Mureb

2015-04-25

Recently an unexpectedly high prevalence of Plasmodium falciparum was found in asymptomatic blood donors living in the southeastern Brazilian Atlantic forest. The bromeliad-malaria paradigm assumes that transmission of Plasmodium vivax and Plasmodium malariae involves species of the subgenus Kerteszia of Anopheles and only a few cases of P. vivax malaria are reported annually in this region. The expectations of this paradigm are a low prevalence of P. vivax and a null prevalence of P. falciparum. Therefore, the aim of this study was to verify if P. falciparum is actively circulating in the southeastern Brazilian Atlantic forest remains. In this study, anophelines were collected with Shannon and CDC-light traps in seven distinct Atlantic forest landscapes over a 4-month period. Field-collected Anopheles mosquitoes were tested by real-time PCR assay in pools of ten, and then each mosquito from every positive pool, separately for P. falciparum and P. vivax. Genomic DNA of P. falciparum or P. vivax from positive anophelines was then amplified by traditional PCR for sequencing of the 18S ribosomal DNA to confirm Plasmodium species. Binomial probabilities were calculated to identify non-random results of the P. falciparum-infected anopheline findings. The overall proportion of anophelines naturally infected with P. falciparum was 4.4% (21/480) and only 0.8% (4/480) with P. vivax. All of the infected mosquitoes were found in intermixed natural and human-modified environments and most were Anopheles cruzii (22/25 = 88%, 18 P. falciparum plus 4 P. vivax). Plasmodium falciparum was confirmed by sequencing in 76% (16/21) of positive mosquitoes, whereas P. vivax was confirmed in only 25% (1/4). Binomial probabilities suggest that P. falciparum actively circulates throughout the region and that there may be a threshold of the forested over human-modified environment ratio upon which the proportion of P. falciparum-infected anophelines increases significantly. These results

In vitro piperaquine susceptibility is not associated with the Plasmodium falciparum chloroquine resistance transporter gene

PubMed Central

2013-01-01

Background Dihydroartemisinin-piperaquine is a new ACT that is administered as single daily dose for three days and has been demonstrated to be tolerated and highly effective for the treatment of uncomplicated Plasmodium falciparum malaria. Piperaquine was used alone to replace chloroquine as the first-line treatment for uncomplicated malaria in China in response to increasing chloroquine resistance in the 1970s. However, the rapid emergence of piperaquine-resistant strains that resulted in the cessation of its use in China in the 1980s, suggests that there is cross-resistance between piperaquine and chloroquine. Very few data are available on cross-resistance between piperaquine and chloroquine, and the data that do exist are often contradictory. Methods In total, 280 P. falciparum isolates, collected between April 2008 and June 2012 from patients hospitalized in France with imported malaria from a malaria-endemic country, were assessed ex vivo for piperaquine and chloroquine susceptibilities by using the standard 42-hour 3H-hypoxanthine uptake inhibition method. The chloroquine resistance-associated mutation K76T in pfcrt was also investigated for the 280 isolates. Results The IC50 for piperaquine ranged from 9.8 nM to 217.3 nM (mean = 81.3 nM. The IC50 for chloroquine ranged from 5.0 nM to 1,918 nM (mean = 83.6 nM. A significant but low correlation was observed between the Log IC50 values for piperaquine and chloroquine (r = 0.145, p < 0.001). However, the coefficient of determination of 0.021 indicates that only 2.1% of the variation in the response to piperaquine is explained by the variation in the response to chloroquine. The mean value for piperaquine was 74.0 nM in the Pfcrt K76 wild-type group (no = 125) and 87.7 nM in the 76 T mutant group (no = 155). This difference was not significant (p = 0.875, Mann Whitney U test). Conclusions The present work demonstrates that there was no cross-resistance between piperaquine and

Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009-2013).

PubMed

Mohon, Abu Naser; Alam, Mohammad Shafiul; Bayih, Abebe Genetu; Folefoc, Asongna; Shahinas, Dea; Haque, Rashidul; Pillai, Dylan R

2014-11-18

Bangladesh is a malaria hypo-endemic country sharing borders with India and Myanmar. Artemisinin combination therapy (ACT) remains successful in Bangladesh. An increase of artemisinin-resistant malaria parasites on the Thai-Cambodia and Thai-Myanmar borders is worrisome. K13 propeller gene (PF3D7_1343700 or PF13_0238) mutations have been linked to both in vitro artemisinin resistance and in vivo slow parasite clearance rates. This group undertook to evaluate if mutations seen in Cambodia have emerged in Bangladesh where ACT use is now standard for a decade. Samples were obtained from Plasmodium falciparum-infected malaria patients from Upazila health complexes (UHC) between 2009 and 2013 in seven endemic districts of Bangladesh. These districts included Khagrachari (Matiranga UHC), Rangamati (Rajasthali UHC), Cox's Bazar (Ramu and Ukhia UHC), Bandarban (Lama UHC), Mymensingh (Haluaghat UHC), Netrokona (Durgapur and Kalmakanda UHC), and Moulvibazar (Sreemangal and Kamalganj UHC). Out of 296 microscopically positive P. falciparum samples, 271 (91.6%) were confirmed as mono-infections by both real-time PCR and nested PCR. The K13 propeller gene from 253 (93.4%) samples was sequenced bi-directionally. One non-synonymous mutation (A578S) was found in Bangladeshi clinical isolates. The A578S mutation was confirmed and lies adjacent to the C580Y mutation, the major mutation causing delayed parasite clearance in Cambodia. Based on computational modeling A578S should have a significant effect on tertiary structure of the protein. The data suggest that P. falciparum in Bangladesh remains free of the C580Y mutation linked to delayed parasite clearance. However, the mutation A578S is present and based on structural analysis could affect K13 gene function. Further in vivo clinical studies are required to validate the effect of this mutation.

Dihydroartemisinin–piperaquine resistance in Plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study

PubMed Central

Amaratunga, Chanaki; Lim, Pharath; Suon, Seila; Sreng, Sokunthea; Mao, Sivanna; Sopha, Chantha; Sam, Baramey; Dek, Dalin; Try, Vorleak; Amato, Roberto; Blessborn, Daniel; Song, Lijiang; Tullo, Gregory S; Fay, Michael P; Anderson, Jennifer M; Tarning, Joel; Fairhurst, Rick M

2016-01-01

Background Artemisinin resistance in Plasmodium falciparum threatens to reduce the efficacy of artemisinin combination therapies (ACTs), thus compromising global efforts to eliminate malaria. Recent treatment failures with dihydroartemisinin-piperaquine, the current first-line ACT in Cambodia, suggest that piperaquine resistance may be emerging in this country. We explored the relation between artemisinin resistance and dihydroartemisinin–piperaquine failures, and sought to confirm the presence of piperaquine-resistant P falciparum infections in Cambodia. Methods In this prospective cohort study, we enrolled patients aged 2–65 years with uncomplicated P falciparum malaria in three Cambodian provinces: Pursat, Preah Vihear, and Ratanakiri. Participants were given standard 3-day courses of dihydroartemisinin–piperaquine. Peripheral blood parasite densities were measured until parasites cleared and then weekly to 63 days. The primary outcome was recrudescent P falciparum parasitaemia within 63 days. We measured piperaquine plasma concentrations at baseline, 7 days, and day of recrudescence. We assessed phenotypic and genotypic markers of drug resistance in parasite isolates. The study is registered with ClinicalTrials.gov, number NCT01736319. Findings Between Sept 4, 2012, and Dec 31, 2013, we enrolled 241 participants. In Pursat, where artemisinin resistance is entrenched, 37 (46%) of 81 patients had parasite recrudescence. In Preah Vihear, where artemisinin resistance is emerging, ten (16%) of 63 patients had recrudescence and in Ratanakiri, where artemisinin resistance is rare, one (2%) of 60 patients did. Patients with recrudescent P falciparum infections were more likely to have detectable piperaquine plasma concentrations at baseline compared with non-recrudescent patients, but did not differ significantly in age, initial parasite density, or piperaquine plasma concentrations at 7 days. Recrudescent parasites had a higher prevalence of kelch13 mutations

In vitro activity of anti-malarial ozonides against an artemisinin-resistant isolate.

PubMed

Baumgärtner, Fabian; Jourdan, Joëlle; Scheurer, Christian; Blasco, Benjamin; Campo, Brice; Mäser, Pascal; Wittlin, Sergio

2017-01-25

Recently published data suggest that artemisinin derivatives and synthetic peroxides, such as the ozonides OZ277 and OZ439, have a similar mode of action. Here the cross-resistance of OZ277 and OZ439 and four additional next-generation ozonides was probed against the artemisinin-resistant clinical isolate Plasmodium falciparum Cam3.I, which carries the K13-propeller mutation R539T (Cam3.I R539T ). The previously described in vitro ring-stage survival assay (RSA 0-3h ) was employed and a simplified variation of the original protocol was developed. At the pharmacologically relevant concentration of 700 nM, all six ozonides were highly effective against the dihydroartemisinin-resistant P. falciparum Cam3.I R539T parasites, showing a per cent survival ranging from <0.01 to 1.83%. A simplified version of the original RSA 0-3h method was developed and gave similar results, thus providing a practical drug discovery tool for further optimization of next-generation anti-malarial peroxides. The absence of in vitro cross-resistance against the artemisinin-resistant clinical isolate Cam3.I R539T suggests that ozonides could be effective against artemisinin-resistant P. falciparum. How this will translate to the human situation in clinical settings remains to be investigated.

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

Inhibition of pyrimidine biosynthesis de novo in Plasmodium falciparum by 2-(4-t-butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone in vitro.

PubMed

Hammond, D J; Burchell, J R; Pudney, M

1985-01-01

The effects of the hydroxynaphthoquinone BW58C on some metabolite levels and the flux of H14CO3 through the de novo pyrimidine biosynthetic pathway of intact Plasmodium falciparum have been studied in vitro using HPLC techniques. 800 nM BW58C appeared to have no significant effect on the energy status of isolated P. falciparum, but at 0.1 nM it caused a dramatic decrease in the concentrations of pyrimidine nucleotides, specifically UTP, during 256 min of incubation. Although about one hour was required to achieve a significant decrease in pyrimidine nucleotide concentrations, a much more rapid inhibition of the flux of H14CO3 through the de novo pathway was found upon addition of 0.1 nM BW58C. This inhibition caused about a 10 fold increase in the radioactivity of carbamoyl-aspartate over a 64 min period, and an overall increase in the concentration of this metabolite of about 3 fold during 256 min of incubation. These effects of BW58C against P. falciparum in vitro are discussed in terms of inhibition of de novo pyrimidine biosynthesis at the site of dihydroorotate dehydrogenase.

Isolation of lactic acid bacteria from Malaysian foods and assessment of the isolates for industrial potential.

PubMed

Mohd Adnan, Ahmad Faris; Tan, Irene K P

2007-05-01

Two traditional fermented food 'tapai' (fermented tapioca) and 'tempoyak' (fermented durian flesh), chilli puree and fresh goat's milk were used as sources for the isolation of lactic acid bacteria (LAB). A total of 126 isolates were obtained and by sequential screening for catalase activity and Gram-staining, 55 were determined to be LAB out of which 16 were established to be homofermentative by the gel plug test. Seven isolates were identified by use of the API 50CHL kit and two lactobacilli strains and one lactococci strain were selected to study their growth and lactic acid production profiles in a time course experiment. The lactobacilli strains, both isolated from 'tapai', produced higher amounts of cells and lactic acid from glucose as compared to the lactococci strain isolated from fresh goat's milk.

Methionine transport in the malaria parasite Plasmodium falciparum.

PubMed

Cobbold, Simon A; Martin, Rowena E; Kirk, Kiaran

2011-01-01

The intraerythrocytic malaria parasite, Plasmodium falciparum, derives amino acids from the digestion of host cell haemoglobin. However, it also takes up amino acids from the extracellular medium. Isoleucine is absent from adult human haemoglobin and an exogenous source of isoleucine is essential for parasite growth. An extracellular source of methionine is also important for the normal growth of at least some parasite strains. In this study we have characterised the uptake of methionine by P. falciparum-infected human erythrocytes, and by parasites functionally isolated from their host cells by saponin-permeabilization of the erythrocyte membrane. Infected erythrocytes take up methionine much faster than uninfected erythrocytes, with the increase attributable to the flux of this amino acid via the New Permeability Pathways induced by the parasite in the erythrocyte membrane. Having entered the infected cell, methionine is taken up by the intracellular parasite via a saturable, temperature-dependent process that is independent of ATP, Na(+) and H(+). Substrate competition studies, and comparison of the transport of methionine with that of isoleucine and leucine, yielded results consistent with the hypothesis that the parasite has at its surface one or more transporters which mediate the flux into and out of the parasite of a broad range of neutral amino acids. These transporters function most efficiently when exchanging one neutral amino acid for another, thus providing a mechanism whereby the parasite is able to import important exogenous amino acids in exchange for surplus neutral amino acids liberated from the digestion of host cell haemoglobin. Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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

Chloroquine-resistant falciparum malaria in Madagascar and Kenya.

PubMed

Aronsson, B; Bengtsson, E; Björkman, A; Pehrson, P O; Rombo, L; Wahlgren, M

1981-08-01

3 African cases of chloroquine-resistent (RI) P. falciparum malaria, proved by recrudescences after administration of recommended doses and adequate serum levels of chloroquine, are described. The 3 patients, Swedish women aged 43, 27, and 41, had never visited areas where chloroquine-resistent P. falciparum is known to exist. 2 patients had taken regular prophylaxis with chloroquine, while the 3rd had interrupted chloroquine use with temporary use of pyrimethamine. All 3 were treated with chloroquine and had serum levels well above those considered necessary for cure of malaria due to sensitive strains of P. falciparum. All had recrudescences, ranging from 13 to 41 days after the previous chloroquine treatment. Reinfection was not possible for any of the patients and none took other drugs during the study period. In 2 cases the Rieckmann in vitro test for resistence failed. In 1 case from Madagascar the in vitro method described by Nguyen-Dinh and Trager produced results indicating resistence and in another case from Madagascar the results indicated probable resistence, but the procedure failed in the case from Kenya. The 2 cases mark the 1st time resistence has been reported from Madagascar. In vivo tests in all cases and in vitro results in 2 cases, together with the adequate serum levels of chloroquine, confirm that malaria due to chloroquine-resistent P. falciparum is being transmitted in some parts of Africa.

Spatio-temporal analysis of the genetic diversity and complexity of Plasmodium falciparum infections in Kedougou, southeastern Senegal.

PubMed

Niang, Makhtar; Thiam, Laty G; Loucoubar, Cheikh; Sow, Abdourahmane; Sadio, Bacary D; Diallo, Mawlouth; Sall, Amadou A; Toure-Balde, Aissatou

2017-01-19

. falciparum isolates. However, mean MOI varied with time of sera collection and decreased over the course of the study (July 2009 to July 2013). This suggests a slow progressive decrease of malaria transmission intensity in Kedougou Region despite the limited impact of preventive and control measures implemented by the National Malaria Control Programme on malaria morbidity and mortality.

Adherence to human lung microvascular endothelial cells (HMVEC-L) of Plasmodium vivax isolates from Colombia

PubMed Central

2013-01-01

Background For years Plasmodium vivax has been considered the cause of benign malaria. Nevertheless, it has been observed that this parasite can produce a severe disease comparable to Plasmodium falciparum. It has been suggested that some physiopathogenic processes might be shared by these two species, such as cytoadherence. Recently, it has been demonstrated that P. vivax-infected erythrocytes (Pv-iEs) have the capacity to adhere to endothelial cells, in which intercellular adhesion molecule-1 (ICAM-1) seems to be involved in this process. Methods Adherence capacity of 21 Colombian isolates, from patients with P. vivax mono-infection to a microvascular line of human lung endothelium (HMVEC-L) was assessed in static conditions and binding was evaluated at basal levels or in tumor necrosis factor (TNF) stimulated cells. The adherence specificity for the ICAM-1 receptor was determined through inhibition with an anti-CD54 monoclonal antibody. Results The majority of P. vivax isolates, 13 out of 21 (61.9%), adhered to the HMVEC-L cells, but P. vivax adherence was at least seven times lower when compared to the four P. falciparum isolates. Moreover, HMVEC-L stimulation with TNF led to an increase of 1.6-fold in P. vivax cytoadhesion, similar to P. falciparum isolates (1.8-fold) at comparable conditions. Also, blockage of ICAM-1 receptor with specific antibodies showed a significant 50% adherence reduction. Conclusions Plasmodium vivax isolates found in Colombia are also capable of adhering specifically in vitro to lung endothelial cells, via ICAM-1 cell receptor, both at basal state and after cell stimulation with TNF. Collectively, these findings reinforce the concept of cytoadherence for P. vivax, but here, to a different endothelial cell line and using geographical distinct isolates, thus contributing to understanding P. vivax biology. PMID:24080027

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

PubMed

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

2016-01-01

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). 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. 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, Pfmdr186Y and 184F 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 presence of mutations in the

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

In vitro growth of Plasmodium falciparum in neonatal blood.

PubMed

Sauerzopf, Ulrich; Honkpehedji, Yabo J; Adgenika, Ayôla A; Feugap, Elianne N; Ngoma, Ghyslain Mombo; Mackanga, Jean-Rodolphe; Lötsch, Felix; Loembe, Marguerite M; Kremsner, Peter G; Mordmüller, Benjamin; Ramharter, Michael

2014-11-18

Children below the age of six months suffer less often from malaria than older children in sub-Saharan Africa. This observation is commonly attributed to the persistence of foetal haemoglobin (HbF), which is considered not to permit growth of Plasmodium falciparum and therefore providing protection against malaria. Since this concept has recently been challenged, this study evaluated the effect of HbF erythrocytes and maternal plasma on in vitro parasite growth of P. falciparum in Central African Gabon. Umbilical cord blood and peripheral maternal blood were collected at delivery at the Albert Schweitzer Hospital in Gabon. Respective erythrocyte suspension and plasma were used in parallel for in vitro culture. In vitro growth rates were compared between cultures supplemented with either maternal or cord erythrocytes. Plasma of maternal blood and cord blood was evaluated. Parasite growth rates were assessed by the standard HRP2-assay evaluating the increase of HRP2 concentration in Plasmodium culture. Culture of P. falciparum using foetal erythrocytes led to comparable growth rates (mean growth rate = 4.2, 95% CI: 3.5 - 5.0) as cultures with maternal red blood cells (mean growth rate =4.2, 95% CI: 3.4 - 5.0) and those from non-malaria exposed individuals (mean growth rate = 4.6, 95% CI: 3.8 - 5.5). Standard in vitro culture of P. falciparum supplemented with either maternal or foetal plasma showed both significantly lower growth rates than a positive control using non-malaria exposed donor plasma. These data challenge the concept of HbF serving as intrinsic inhibitor of P. falciparum growth in the first months of life. Erythrocytes containing HbF are equally permissive to P. falciparum growth in vitro. However, addition of maternal and cord plasma led to reduced in vitro growth which may translate to protection against clinical disease or show synergistic effects with HbF in vivo. Further studies are needed to elucidate the pathophysiology of innate and acquired

Identification of O-GlcNAcylated proteins in Plasmodium falciparum.

PubMed

Kupferschmid, Mattis; Aquino-Gil, Moyira Osny; Shams-Eldin, Hosam; Schmidt, Jörg; Yamakawa, Nao; Krzewinski, Frédéric; Schwarz, Ralph T; Lefebvre, Tony

2017-11-29

Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans. The P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS). While low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins. This study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle.

Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

PubMed

Price, Ric N; Uhlemann, Anne-Catrin; Brockman, Alan; McGready, Rose; Ashley, Elizabeth; Phaipun, Lucy; Patel, Rina; Laing, Kenneth; Looareesuwan, Sornchai; White, Nicholas J; Nosten, François; Krishna, Sanjeev

The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with

Identification of Plasmodium falciparum reticulocyte binding protein homologue 5-interacting protein, PfRipr, as a highly conserved blood-stage malaria vaccine candidate.

PubMed

Ntege, Edward H; Arisue, Nobuko; Ito, Daisuke; Hasegawa, Tomoyuki; Palacpac, Nirianne M Q; Egwang, Thomas G; Horii, Toshihiro; Takashima, Eizo; Tsuboi, Takafumi

2016-11-04

Genetic variability in Plasmodium falciparum malaria parasites hampers current malaria vaccine development efforts. Here, we hypothesize that to address the impact of genetic variability on vaccine efficacy in clinical trials, conserved antigen targets should be selected to achieve robust host immunity across multiple falciparum strains. Therefore, suitable vaccine antigens should be assessed for levels of polymorphism and genetic diversity. Using a total of one hundred and two clinical isolates from a region of high malaria transmission in Uganda, we analyzed extent of polymorphism and genetic diversity in four recently reported novel blood-stage malaria vaccine candidate proteins: Rh5 interacting protein (PfRipr), GPI anchored micronemal antigen (PfGAMA), rhoptry-associated leucine zipper-like protein 1 (PfRALP1) and Duffy binding-like merozoite surface protein 1 (PfMSPDBL1). In addition, utilizing the wheat germ cell-free system, we expressed recombinant proteins for the four candidates based on P. falciparum laboratory strain 3D7 sequences, immunized rabbits to obtain specific antibodies (Abs) and performed functional growth inhibition assay (GIA). The GIA activity of the raised Abs was demonstrated using both homologous 3D7 and heterologous FVO strains in vitro. Both pfripr and pfralp1 are less polymorphic but the latter is comparatively more diverse, with varied number of regions having insertions and deletions, asparagine and 6-mer repeats in the coding sequences. Pfgama and pfmspdbl1 are polymorphic and genetically diverse among the isolates with antibodies against the 3D7-based recombinant PfGAMA and PfMSPDBL1 inhibiting merozoite invasion only in the 3D7 but not FVO strain. Moreover, although Abs against the 3D7-based recombinant PfRipr and PfRALP1 proteins potently inhibited merozoite invasion of both 3D7 and FVO, the GIA activity of anti-PfRipr was much higher than that of anti-PfRALP1. Thus, PfRipr is regarded as a promising blood-stage vaccine

P. falciparum Modulates Erythroblast Cell Gene Expression in Signaling and Erythrocyte Production Pathways

PubMed Central

Tamez, Pamela A.; Liu, Hui; Wickrema, Amittha; Haldar, Kasturi

2011-01-01

Global, genomic responses of erythrocytes to infectious agents have been difficult to measure because these cells are e-nucleated. We have previously demonstrated that in vitro matured, nucleated erythroblast cells at the orthochromatic stage can be efficiently infected by the human malaria parasite Plasmodium falciparum. We now show that infection of orthochromatic cells induces change in 609 host genes. 592 of these transcripts are up-regulated and associated with metabolic and chaperone pathways unique to P. falciparum infection, as well as a wide range of signaling pathways that are also induced in related apicomplexan infections of mouse hepatocytes or human fibroblast cells. Our data additionally show that polychromatophilic cells, which precede the orthochromatic stage and are not infected when co-cultured with P. falciparum, up-regulate a small set of genes, at least two of which are associated with pathways of hematopoiesis and/or erythroid cell development. These data support the idea that P. falciparum affects erythropoiesis at multiple stages during erythroblast differentiation. Further P. falciparum may modulate gene expression in bystander erythroblasts and thus influence pathways of erythrocyte development. This study provides a benchmark of the host erythroblast cell response to infection by P. falciparum. PMID:21573240

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

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.

Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru

PubMed Central

Akinyi, Sheila; Hayden, Tonya; Gamboa, Dionicia; Torres, Katherine; Bendezu, Jorge; Abdallah, Joseph F.; Griffing, Sean M.; Quezada, Wilmer Marquiño; Arrospide, Nancy; De Oliveira, Alexandre Macedo; Lucas, Carmen; Magill, Alan J.; Bacon, David J.; Barnwell, John W.; Udhayakumar, Venkatachalam

2013-01-01

The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times. PMID:24077522

Chemical genetics of Plasmodium falciparum

PubMed Central

Guiguemde, W. Armand; Shelat, Anang A.; Bouck, David; Duffy, Sandra; Crowther, Gregory J.; Davis, Paul H.; Smithson, David C.; Connelly, Michele; Clark, Julie; Zhu, Fangyi; Jiménez-Díaz, María B; Martinez, María S; Wilson, Emily B.; Tripathi, Abhai K.; Gut, Jiri; Sharlow, Elizabeth R.; Bathurst, Ian; El Mazouni, Farah; Fowble, Joseph W; Forquer, Isaac; McGinley, Paula L; Castro, Steve; Angulo-Barturen, Iñigo; Ferrer, Santiago; Rosenthal, Philip J.; DeRisi, Joseph L; Sullivan, David J.; Lazo, John S.; Roos, David S.; Riscoe, Michael K.; Phillips, Margaret A.; Rathod, Pradipsinh K.; Van Voorhis, Wesley C.; Avery, Vicky M; Guy, R. Kiplin

2010-01-01

Malaria caused by Plasmodium falciparum is a catastrophic disease worldwide (880,000 deaths yearly). Vaccine development has proved difficult and resistance has emerged for most antimalarials. In order to discover new antimalarial chemotypes, we have employed a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library, many of which exhibited potent in vitro activity against drug resistant strains, and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in multiple organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Overall, our findings provide the scientific community with new starting points for malaria drug discovery. PMID:20485428

Plasmodium falciparum uses vitamin E to avoid oxidative stress.

PubMed

Sussmann, Rodrigo A C; Fotoran, Wesley L; Kimura, Emilia A; Katzin, Alejandro M

2017-10-10

Plasmodium falciparum is sensitive to oxidative stress in vitro and in vivo, and many drugs such as artemisinin, chloroquine and cercosporin interfere in the parasite's redox system. To minimize the damage caused by reactive radicals, antioxidant enzymes and their substrates found in parasites and in erythrocytes must be functionally active. It was shown that P. falciparum synthesizes vitamin E and that usnic acid acts as an inhibitor of its biosynthesis. Vitamin E is a potent antioxidant that protects polyunsaturated fatty acids from lipid peroxidation, and this activity can be measured by detecting its oxidized product and by evaluating reactive oxygen species (ROS) levels. Here, we demonstrated that ROS levels increased in P. falciparum when vitamin E biosynthesis was inhibited by usnic acid treatment and decreased to basal levels if exogenous vitamin E was added. Furthermore, we used metabolic labelling to demonstrate that vitamin E biosynthesized by the parasite acts as an antioxidant since we could detect its radiolabeled oxidized product. The treatment with chloroquine or cercosporin of the parasites increased the ratio between α-tocopherolquinone and α-tocopherol. Our findings demonstrate that vitamin E produced endogenously by P. falciparum is active as an antioxidant, probably protecting the parasite from the radicals generated by drugs.

21 CFR 101.95 - “Fresh,” “freshly frozen,” “fresh frozen,” “frozen fresh.”

Code of Federal Regulations, 2010 CFR

2010-04-01

... fresh.â 101.95 Section 101.95 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... Descriptive Claims That Are Neither Nutrient Content Claims nor Health Claims § 101.95 “Fresh,” “freshly... frozen by a freezing system such as blast-freezing (sub-zero Fahrenheit temperature with fast moving air...

Efficacy of chloroquine for the treatment of uncomplicated Plasmodium falciparum malaria in Honduras.

PubMed

Mejia Torres, Rosa Elena; 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

2013-05-01

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.

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

Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ting, L; Shi, W; Lewandowicz, A

Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of the polyamine pathway are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potentmore » malaria-specific inhibitors of these enzymes have not previously been described. 5'-methylthio-Immucillin-H, a transition state analogue inhibitor that is selective for malarial over human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may have application as antimalarials.« less

Floricoccus tropicus gen. nov., sp. nov. and Floricoccus penangensis sp. nov. isolated from fresh flowers of durian tree and hibiscus.

PubMed

Chuah, Li-Oon; Yap, Kien-Pong; Shamila-Syuhada, Ahamed Kamal; Thong, Kwai Lin; Ahmad, Rosma; Liong, Min Tze; Rusul, Gulam

2017-12-01

Three strains of Gram-staining-positive, coccus-shaped, lactic acid bacteria, designated as HibF3 T , HibF2 and HibF5 were isolated from fresh flowers of hibiscus, and a fourth, DF1 T , was isolated from fresh flowers of durian tree, in Penang, Malaysia. Taxonomic characterisation was performed by polyphasic analysis. Sequence similarities of the 16S rRNA gene and the housekeeping rpoA and pheS genes of these strains with their closely-related lactococcal and streptococcal relatives were 92-94, 78 and 81 %, respectively. The results of phylogenetic analysis indicated that strains DF1 T , HibF2, HibF5 and HibF3 T were clustered together but were clearly separated from species of the genera Streptococcus and Lactococcus, indicating that they represent members of a novel genus of the family Streptococcaceae. Calculation of average nucleotide identity (ANI) values between the genomes of DF1 T and HibF3 T yielded values of 92.50-92.93 %. ANI values below the cut-off value and distinctive chemotaxonomic characteristics supported the hypothesis that these strains represented two novel species. Major cellular fatty acids in DF1 T , HibF2 and HibF5 were C18 : 1ω7c and C16 : 0, while C12 : 0 and C14 : 0 were also dominant, in addition to C18 : 1ω7c and C16 : 0, in HibF3 T . A novel genus is proposed with the name Floricoccus gen. nov. which consists of two species, Floricoccus tropicus sp. nov as the type species, and Floricoccus penangensis sp. nov. The respective type strains are DF1 T (=LMG 29833 T =JCM 31733 T ) and HibF3 T (=LMG 29831 T =DSM 31735 T ).

Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number

PubMed Central

Brockman, Alan; McGready, Rose; Ashley, Elizabeth; Phaipun, Lucy; Patel, Rina; Laing, Kenneth; Looareesuwan, Sornchai; White, Nicholas J; Nosten, François; Krishna, Sanjeev

2015-01-01

Summary Background The borders of Thailand harbour the world’s most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. Methods The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Findings Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6·3 (95% CI 2·9–13·8, p<0·001) after mefloquine monotherapy and 5·4 (2·0-14·6, p=0·001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Interpretation Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Relevance to practice Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a

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.

Study on E. coli and Salmonella biofilms from fresh fruits and vegetables.

PubMed

Amrutha, Balagopal; Sundar, Kothandapani; Shetty, Prathapkumar Halady

2017-04-01

Foodborne outbreaks associated with fresh fruits and vegetables are on the rise worldwide. Biofilm formation is one of the important traits of pathogens making them strongly attached to substrates as well as express virulence phenotypes. Present study investigates the biofilm forming ability of E. coli and Salmonella sp. isolated from fresh fruits and vegetables. A total of 53 strains, including 35 E. coli and 18 Salmonella sp. isolated from different fruit and vegetable samples were taken into account for the study. Initial screening for biofilm formation was done using Congo Red agar plate test. Results revealed that 22.8% E. coli and 22.2% Salmonella sp. were potential biofilm formers. However, the MTP (Micro-Titre Plate) assay suggested more isolates of both E. coli and Salmonella sp. were moderate to strong biofilm producers. Agar plate diffusion assay with Agrobacterium tumefaciens NTL-4 showed the production of quorum signaling molecules (AHLs) by three isolates of E. coli and one Salmonella sp. Two E. coli isolates showed a significant amount of EPS production indicating higher biofilm forming potential. The Presence of LUX R homologue gene ( sdi A) in two of the Salmonella isolates were confirmed by PCR which demonstrated their potential pathogenicity. Results of the work underline the biofilm forming and potentially virulent capacities of isolates from the surface of fruits and vegetables.

Insights into the Performance of SD Bioline Malaria Ag P.f/Pan Rapid Diagnostic Test and Plasmodium falciparum Histidine-Rich Protein 2 Gene Variation in Madagascar.

PubMed

Willie, Nigani; Mehlotra, Rajeev K; Howes, Rosalind E; Rakotomanga, Tovonahary A; Ramboarina, Stephanie; Ratsimbasoa, Arsène C; Zimmerman, Peter A

2018-06-01

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) forms the basis of many current malaria rapid diagnostic tests (RDTs). However, the parasites lacking part or all of the pfhrp2 gene do not express the PfHRP2 protein and are, therefore, not identifiable by PfHRP2-detecting RDTs. We evaluated the performance of the SD Bioline Malaria Ag P.f/Pan RDT together with pfhrp2 variation in Madagascar. Genomic DNA isolated from 260 patient blood samples were polymerase chain reaction (PCR)-amplified for the parasite 18S rRNA and pfhrp2 genes. Post-PCR ligation detection reaction-fluorescent microsphere assay (LDR-FMA) was performed for the identification of parasite species. Plasmodium falciparum histidine-rich protein 2 amplicons were sequenced. Polymerase chain reaction diagnosis of patient samples showed that 29% (75/260) were infected and P. falciparum was present in 95% (71/75) of these PCR-positive samples. Comparing RDT and P. falciparum detection by LDR-FMA, eight samples were RDT negative but P. falciparum positive (false negatives), all of which were pfhrp2 positive. The sensitivity and specificity of the RDT were 87% and 90%, respectively. Seventy-three samples were amplified for pfhrp2 , from which nine randomly selected amplicons were sequenced, yielding 13 sequences. Amplification of pfhrp2 , combined with RDT analysis and P. falciparum detection by LDR-FMA, showed that there was no indication of pfhrp2 deletion. Sequence analysis of pfhrp2 showed that the correlation between pfhrp2 sequence structure and RDT detection rates was unclear. Although the observed absence of pfhrp2 deletion from the samples screened here is encouraging, continued monitoring of the efficacy of the SD Bioline Malaria Ag P.f/Pan RDT for malaria diagnosis in Madagascar is warranted.

Plasmodium falciparum: attenuation by irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 tomore » 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.« less

The Plasmodium gaboni genome illuminates allelic dimorphism of immunologically important surface antigens in P. falciparum.

PubMed

Roy, Scott William

2015-12-01

In the deadly human malaria parasite Plasmodium falciparum, several major merozoite surface proteins (MSPs) show a striking pattern of allelic diversity called allelic dimorphism (AD). In AD, the vast majority of observed alleles fall into two highly divergent allelic classes, with recombinant alleles being rare or not observed, presumably due to repression by natural selection (recombination suppression, or RS). The three AD loci, merozoite surface proteins (MSPs) 1, 2, and 6, along with MSP3, which also exhibits RS among four allelic classes, can be collectively called AD/RS. The causes of AD/RS and the evolutionary history of allelic diversity at these loci remain mysterious. The few available sequences from a single closely related chimpanzee parasite, P. reichenowi, have suggested that for 3/4 loci, AD/RS is an ancient state that has been retained in P. falciparum since well before the P. falciparum-P. reichenowi ancestor. On the other hand, based on comparative sequence analysis, we recently suggested that (i) AD/RS P. falciparum loci have undergone interallelic recombination over longer evolutionary times (on the timescale of recent speciation events), and thus (ii) AD/RS may be a recent phenomenon. The recent publication of genomic sequencing efforts for P. gaboni, an outgroup to P. falciparum and P. reichenowi, allows for improved reconstruction of the evolutionary history of these loci. In this work, I report genic sequence for P. gaboni for all four AD/RS P. falciparum loci (MSP1, 2, 3, and 6). Comparison of these sequences with available P. falciparum and P. reichenowi data strengthens the evidence for interallelic recombination over the evolutionary history of these species and also strengthens the case that AD/RS at these loci is ancient. Combined with previous results, these data provide evidence that AD/RS at different loci has evolved at several different times in the evolutionary history of P. falciparum: (i) before the P. gaboni-P. falciparum

Origin of the human malaria parasite Plasmodium falciparum in gorillas

PubMed Central

Liu, Weimin; Li, Yingying; Learn, Gerald H.; Rudicell, Rebecca S.; Robertson, Joel D.; Keele, Brandon F.; Ndjango, Jean-Bosco N.; Sanz, Crickette M.; Morgan, David B.; Locatelli, Sabrina; Gonder, Mary K.; Kranzusch, Philip J.; Walsh, Peter D.; Delaporte, Eric; Mpoudi-Ngole, Eitel; Georgiev, Alexander V.; Muller, Martin N.; Shaw, George M.; Peeters, Martine; Sharp, Paul M.; Rayner, Julian C.; Hahn, Beatrice H.

2010-01-01

Plasmodium falciparum is the most prevalent and lethal of the malaria parasites infecting humans, yet the origin and evolutionary history of this important pathogen remain controversial. Here, we developed a novel polymerase chain reaction based single genome amplification strategy to identify and characterize Plasmodium spp. DNA sequences in fecal samples of wild-living apes. Among nearly 3,000 specimens collected from field sites throughout central Africa, we found Plasmodium infection in chimpanzees (Pan troglodytes) and western gorillas (Gorilla gorilla), but not in eastern gorillas (Gorilla beringei) or bonobos (Pan paniscus). Ape plasmodial infections were highly prevalent, widely distributed, and almost always comprised of mixed parasite species. Analysis of more than 1,100 mitochondrial, apicoplast and nuclear gene sequences from chimpanzees and gorillas revealed that 99% grouped within one of six host-specific lineages representing distinct Plasmodium species within the subgenus Laverania. One of these from western gorillas was comprised of parasites that were nearly identical to P. falciparum. In phylogenetic analyses of full-length mitochondrial sequences, human P. falciparum formed a monophyletic lineage within the gorilla parasite radiation. These findings indicate that P. falciparum is of gorilla and not of chimpanzee, bonobo or ancient human origin. PMID:20864995

Plasmodium falciparum: a simplified technique for obtaining singly infected erythrocytes.

PubMed

Puthia, Manoj K; Tan, Kevin S W

2005-02-01

We report the development of a simple technique involving 15 ml polypropylene tubes and a rotatory incubator for obtaining erythrocytes singly infected with Plasmodium falciparum. This technique will be useful for cloning of the parasite. Our finding that P. falciparum merozoite invasion is inhibited during rotation suggests that this method may also be useful for the study of parasite-erythrocyte interactions under dynamic circulatory conditions.

Antimalarial Activity of KAF156 in Falciparum and Vivax Malaria.

PubMed

White, Nicholas J; Duong, Tran T; Uthaisin, Chirapong; Nosten, François; Phyo, Aung P; Hanboonkunupakarn, Borimas; Pukrittayakamee, Sasithon; Jittamala, Podjanee; Chuthasmit, Kittiphum; Cheung, Ming S; Feng, Yiyan; Li, Ruobing; Magnusson, Baldur; Sultan, Marc; Wieser, Daniela; Xun, Xiaolei; Zhao, Rong; Diagana, Thierry T; Pertel, Peter; Leong, F Joel

2016-09-22

KAF156 belongs to a new class of antimalarial agents (imidazolopiperazines), with activity against asexual and sexual blood stages and the preerythrocytic liver stages of malarial parasites. We conducted a phase 2, open-label, two-part study at five centers in Thailand and Vietnam to assess the antimalarial efficacy, safety, and pharmacokinetic profile of KAF156 in adults with acute Plasmodium vivax or P. falciparum malaria. Assessment of parasite clearance rates in cohorts of patients with vivax or falciparum malaria who were treated with multiple doses (400 mg once daily for 3 days) was followed by assessment of the cure rate at 28 days in a separate cohort of patients with falciparum malaria who received a single dose (800 mg). Median parasite clearance times were 45 hours (interquartile range, 42 to 48) in 10 patients with falciparum malaria and 24 hours (interquartile range, 20 to 30) in 10 patients with vivax malaria after treatment with the multiple-dose regimen and 49 hours (interquartile range, 42 to 54) in 21 patients with falciparum malaria after treatment with the single dose. Among the 21 patients who received the single dose and were followed for 28 days, 1 had reinfection and 7 had recrudescent infections (cure rate, 67%; 95% credible interval, 46 to 84). The mean (±SD) KAF156 terminal elimination half-life was 44.1±8.9 hours. There were no serious adverse events in this small study. The most common adverse events included sinus bradycardia, thrombocytopenia, hypokalemia, anemia, and hyperbilirubinemia. Vomiting of grade 2 or higher occurred in 2 patients, 1 of whom discontinued treatment because of repeated vomiting after receiving the single 800-mg dose. More adverse events were reported in the single-dose cohort, which had longer follow-up, than in the multiple-dose cohorts. KAF156 showed antimalarial activity without evident safety concerns in a small number of adults with uncomplicated P. vivax or P. falciparum malaria. (Funded by Novartis and

Inflammatory reactions in placental blood of Plasmodium falciparum-infected women and high concentrations of soluble E-selectin and a circulating P. falciparum protein in the cord sera.

PubMed Central

Jakobsen, P H; Rasheed, F N; Bulmer, J N; Theisen, M; Ridley, R G; Greenwood, B M

1998-01-01

To better understand reasons for increased susceptibility to malaria in pregnancy; and the interrelationships between maternal malaria, local immune reactions and the development of the fetus, concentrations of soluble interleukin-10 (IL-10), cytokine receptors, adhesion molecules, a Plasmodium falciparum protein, glutamate-rich protein (GLURP) and antibodies to P. falciparum rhoptry-associated protein-1 were measured among 105 Gambian women and their neonates. Peripheral blood concentrations of IL-10, soluble cytokine receptors and soluble adhesion molecules were found to be different from those concentrations measured in the placenta. Markers of inflammatory reactions: IL-10, sIL-2R, sIL-4R, and soluble tumour necrosis factor receptor I (sTNF-RI) were found in high concentrations in the placenta, indicating that inflammatory reactions take place in the placenta which has been regarded as an immunoprivileged site. Concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intracellular adhesion molecule-1 (sICAM-1), potential adhesion receptors for malaria parasites, were associated with an active P. falciparum infection in the placenta although the associations did not reach significance. P. falciparum exoantigen, GLURP, was detected in cord blood indicating transplacental passage of malarial antigens. Concentrations of E-selectin were higher in cord blood samples compared with peripheral blood samples. This appeared to be associated with development of cord endothelial cells and not with P. falciparum infection. PMID:9616377

Pernicious plans revealed: Plasmodium falciparum genome wide expression analysis.

PubMed

Llinás, Manuel; DeRisi, Joseph L

2004-08-01

The asexual intraerythrocytic developmental cycle (IDC) of Plasmodium falciparum is responsible for the majority of the clinical manifestations of malaria in humans. Although malaria has been studied for over a century, the elucidation of the full genome sequence of P. falciparum has now allowed for in-depth studies of gene expression throughout the entire intraerythrocytic stage. As the mainstays of anti-malarial chemotherapy become increasingly ineffective, we need a deeper understanding of fundamental plasmodial bioregulatory mechanisms to successfully subvert them. Recent gene expression studies have begun to examine different aspects of the IDC and are providing key insights into the basic mechanisms of Plasmodium gene regulation and are helping to define gene functions. However, to date, no transcription factor has been fully characterized from Plasmodium and the definitive identification of cis-acting regulatory elements along with their corresponding trans-acting partners is still lacking. The characterization of the transcriptome of P. falciparum is the first major step towards the understanding of the genome wide regulation of gene expression in this parasite. IDC expression data for almost every gene in the P. falciparum genome can now be publicly queried at and. The results of these studies suggest promising leads for identifying novel targets for anti-malarial therapeutics and vaccines in addition to providing a solid foundation for the ongoing elucidation of plasmodial gene expression.

Longitudinal study assessing the return of chloroquine susceptibility of Plasmodium falciparum in isolates from travellers returning from West and Central Africa, 2000-2011.

PubMed

Gharbi, Myriam; Flegg, Jennifer A; Hubert, Véronique; Kendjo, Eric; Metcalf, Jessica E; Bertaux, Lionel; Guérin, Philippe J; Le Bras, Jacques; Aboubaca, Ahmed; Agnamey, Patrice; Angoulvant, Adela; Barbut, Patricia; Basset, Didier; Belkadi, Ghania; Bellanger, Anne Pauline; Bemba, Dieudonné; Benoit-Vica, Françoise; Berry, Antoine; Bigel, Marie-Laure; Bonhomme, Julie; Botterel, Françoise; Bouchaud, Olivier; Bougnoux, Marie-Elisabeth; Bourée, Patrice; Bourgeois, Nathalie; Branger, Catherine; Bret, Laurent; Buret, Bernadette; Casalino, Enrique; Chevrier, Sylviane; Conquere de Monbrison, Frédérique; Cuisenier, Bernadette; Danis, Martin; Darde, Marie-Laure; De Gentile, Ludovic; Delarbre, Jean-Marie; Delaunay, Pascal; Delaval, Anne; Desoubeaux, Guillaume; Develoux, Michel; Dunand, Jean; Durand, Rémy; Eloy, Odile; Fauchet, Nathalie; Faugere, Bernard; Faye, Alber; Fenneteau, Odile; Flori, Pierre; Fontrouge, Madeleine; Garabedian, Chantal; Gayandrieu, Françoise; Godineau, Nadine; Houzé, Pascal; Houzé, Sandrine; Hurst, Jean-Pierre; Ichou, Houria; Lachaud, Laurence; Lebuisson, Agathe; Lefevre, Magalie; LeGuern, Anne-Sophie; Le Moal, Gwenaë; Lusina, Daniel; Machouart, Marie-Claude; Malvy, Denis; Matheron, Sophie; Maubon, Danièle; Mechali, Denis; Megarbane, Bruno; Menard, Guillaume; Millon, Laurence; Aiach, Muriel Mimoun; Minodier, Philippe; Morelle, Christelle; Nevez, Gilles; Parola, Philippe; Parzy, Daniel; Patey, Olivier; Patoz, Pierre; Penn, Pascale; Perignon, Alice; Picot, Stéphane; Pilo, Jean-Etienne; Poilane, Isabelle; Pons, Denis; Poupart, Marie; Pradines, Bruno; Raffenot, Didier; Rapp, Christophe; Receveur, Marie-Catherine; Sarfati, Claudine; Senghor, Yaye; Simon, Fabrice; Siriez, Jean-Yves; Taudon, Nicolas; Thellier, Marc; Thouvenin, Maxime; Toubas, Dominique

2013-01-25

Chloroquine (CQ) was the main malaria therapy worldwide from the 1940s until the 1990s. Following the emergence of CQ-resistant Plasmodium falciparum, most African countries discontinued the use of CQ, and now promote artemisinin-based combination therapy as the first-line treatment. This change was generally initiated during the last decade in West and Central Africa. The aim of this study is to describe the changes in CQ susceptibility in this African region, using travellers returning from this region as a sentinel system. The study was conducted by the Malaria National Reference Centre, France. The database collated the pfcrtK76T molecular marker for CQ susceptibility and the in vitro response to CQ of parasites from travellers' isolates returning from Senegal, Mali, Ivory Coast or Cameroon. As a proxy of drug pressure, data regarding CQ intake in febrile children were collated for the study period. Logistic regression models were used to detect trends in the proportions of CQ resistant isolates. A total of 2874 parasite isolates were genotyped between 2000-2011. The prevalence of the pfcrt76T mutant genotype significantly decreased for Senegal (from 78% to 47%), Ivory Coast (from 63% to 37%), Cameroon (from 90% to 59%) and remained stable for Mali. The geometric mean of the 50% inhibitory concentration (IC50) of CQ in vitro susceptibility and the proportion of resistant isolates (defining resistance as an IC50 value > 100 nM) significantly decreased for Senegal (from 86 nM (59%) to 39 nM (25%)), Mali (from 84 nM (50%) to 51 nM (31%)), Ivory Coast (from 75 nM (59%) to 29 nM (16%)) and Cameroon (from 181 nM (75%) to 51 nM (37%)). Both analyses (molecular and in vitro susceptibility) were performed for the 2004-2011 period, after the four countries had officially discontinued CQ and showed an accelerated decline of the resistant isolates for the four countries. Meanwhile, CQ use among children significantly deceased in this region (fixed effects slope�

The effect of sulphadoxine/pyrimethamine on gametocytes in falciparum malaria.

PubMed

Ittiravivongs, A; Vasuvat, C; Kongrod, S

1984-09-01

A study of the effect of sulphadoxine/pyrimethamine (Fansidar) on P. falciparum's gametocytes in peripheral blood was carried out in Western Thailand. One group of 77 patients with asexual form P. falciparum sensitive to Fansidar were followed weekly to detect the appearance and the duration of gametocytes in peripheral blood after Fansidar treatment on the basis of thick blood film examination. Another group of 14 patients with sexual form P. falciparum was not given any antimalarial treatment and also followed up weekly. No significant difference of average duration of detectable gametocytes was observed between the groups. The average number of days that gametocytes appeared after asexual form in patients receiving treatment was the same as in the untreated group. It is unlikely that Fansidar has the stimulating effect on gametocytogenesis as previously reported.

Glucose-6-phosphate metabolism in Plasmodium falciparum.

PubMed

Preuss, Janina; Jortzik, Esther; Becker, Katja

2012-07-01

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based. Copyright © 2012 Wiley Periodicals, Inc.

The efficiency of sporozoite transmission in the human malarias, Plasmodium falciparum and P. vivax*

PubMed Central

Burkot, T. R.; Graves, P. M.; Cattan, J. A.; Wirtz, R. A.; Gibson, F. D.

1987-01-01

Reported are malaria sporozoite and inoculation rates over a 1-year period in eight epidemiologically defined villages of different endemicity in Madang Province, Papua New Guinea. In the study, more than 41 000 wild-caught mosquitos were analysed for Plasmodium falciparum and P. vivax sporozoites by ELISA. In a given village the entomological inoculation rates correlated strongly with the prevalences of both these malarial parasites in children. However, the prevalence of P. falciparum infections in children was much higher than that of P. vivax, despite similar inoculation rates for the two species. These data suggest that in Papua New Guinea P. falciparum is more efficiently transmitted than P. vivax from mosquito to man. The increased efficiency of transmission of P. falciparum may be due to the heavier sporozoite densities in wild-caught mosquitos naturally infected with P. falciparum sporozoites that were tenfold greater than the sporozoite densities in mosquitos infected with P. vivax. PMID:3311441

Insight into k13-propeller gene polymorphism and ex vivo DHA-response profiles from Cameroonian isolates.

PubMed

Menard, Sandie; Tchoufack, Joëlle Njila; Maffo, Christelle Ngou; Nsango, Sandrine E; Iriart, Xavier; Abate, Luc; Tsapi, Majoline Tchioffo; Awono-Ambéné, Parfait H; Abega Mekongo, Francis A; Morlais, Isabelle; Berry, Antoine

2016-11-26

The spread of Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia is a major source of concern and the emergence of resistance in Africa would have dramatic consequences, by increasing malaria mortality and morbidity. It is therefore urgent to implement regular monitoring in sentinel sites in sub-Saharan Africa using robust and easy-to-implement tools. The prevalence of k13-propeller mutations and the phenotypic profiles are poorly known in sub-Saharan Africa. Here, the k13-propeller polymorphism was compared to both ex vivo susceptibility to DHA and early parasitological and clinical responses to artemisinin combination therapy (ACT). Plasmodium falciparum isolates were collected in 2015 in Yaoundé (Cameroon) from patients treated with dihydroartemisinin-piperaquine combination. Samples were analysed for their susceptibility to artemisinin using the k13-propeller sequencing, the ex vivo ring-stage survival assay, the in vivo parasite positive rate and the clinical statute at day 2. None of the collected isolates revealed the presence of resistance mutations in the k13-propeller sequence. The median ring-stage survival rate for all the 64 interpretable isolates after a 6-hour pulse of 700 nM dihydroartemisinin was low, 0.49% (IQR: 0-1.3). Total parasite clearance was observed for 87.5% of patients and the remaining parasitaemic isolates (12.5%) showed a high reduction of parasite load, ranging from 97.5 to 99.9%. Clinical symptoms disappeared in 92.8% of cases. This study demonstrated the absence of k13-resistant genotypes in P. falciparum isolates from Cameroon. Only synonymous mutations were found with a low prevalence (4.3%). A good association between k13 genotypes and the ex vivo ring-stage survival assay or parasitological and clinical data was obtained. These results give a baseline for the long-term monitoring of artemisinin derivative efficacy in Africa.

RTS,S/AS01 malaria vaccine mismatch observed among Plasmodium falciparum isolates from southern and central Africa and globally.

PubMed

Pringle, Julia C; Carpi, Giovanna; Almagro-Garcia, Jacob; Zhu, Sha Joe; Kobayashi, Tamaki; Mulenga, Modest; Bobanga, Thierry; Chaponda, Mike; Moss, William J; Norris, Douglas E

2018-04-26

The RTS,S/AS01 malaria vaccine encompasses the central repeats and C-terminal of Plasmodium falciparum circumsporozoite protein (PfCSP). Although no Phase II clinical trial studies observed evidence of strain-specific immunity, recent studies show a decrease in vaccine efficacy against non-vaccine strain parasites. In light of goals to reduce malaria morbidity, anticipating the effectiveness of RTS,S/AS01 is critical to planning widespread vaccine introduction. We deep sequenced C-terminal Pfcsp from 77 individuals living along the international border in Luapula Province, Zambia and Haut-Katanga Province, the Democratic Republic of the Congo (DRC) and compared translated amino acid haplotypes to the 3D7 vaccine strain. Only 5.2% of the 193 PfCSP sequences from the Zambia-DRC border region matched 3D7 at all 84 amino acids. To further contextualize the genetic diversity sampled in this study with global PfCSP diversity, we analyzed an additional 3,809 Pfcsp sequences from the Pf3k database and constructed a haplotype network representing 15 countries from Africa and Asia. The diversity observed in our samples was similar to the diversity observed in the global haplotype network. These observations underscore the need for additional research assessing genetic diversity in P. falciparum and the impact of PfCSP diversity on RTS,S/AS01 efficacy.

Electrostatic channeling in P. falciparum DHFR-TS: Brownian dynamics and Smoluchowski modeling.

PubMed

Metzger, Vincent T; Eun, Changsun; Kekenes-Huskey, Peter M; Huber, Gary; McCammon, J Andrew

2014-11-18

We perform Brownian dynamics simulations and Smoluchowski continuum modeling of the bifunctional Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (P. falciparum DHFR-TS) with the objective of understanding the electrostatic channeling of dihydrofolate generated at the TS active site to the DHFR active site. The results of Brownian dynamics simulations and Smoluchowski continuum modeling suggest that compared to Leishmania major DHFR-TS, P. falciparum DHFR-TS has a lower but significant electrostatic-mediated channeling efficiency (?15-25%) at physiological pH (7.0) and ionic strength (150 mM). We also find that removing the electric charges from key basic residues located between the DHFR and TS active sites significantly reduces the channeling efficiency of P. falciparum DHFR-TS. Although several protozoan DHFR-TS enzymes are known to have similar tertiary and quaternary structure, subtle differences in structure, active-site geometry, and charge distribution appear to influence both electrostatic-mediated and proximity-based substrate channeling.

More than just immune evasion: Hijacking complement by Plasmodium falciparum.

PubMed

Schmidt, Christoph Q; Kennedy, Alexander T; Tham, Wai-Hong

2015-09-01

Malaria remains one of the world's deadliest diseases. Plasmodium falciparum is responsible for the most severe and lethal form of human malaria. P. falciparum's life cycle involves two obligate hosts: human and mosquito. From initial entry into these hosts, malaria parasites face the onslaught of the first line of host defence, the complement system. In this review, we discuss the complex interaction between complement and malaria infection in terms of hosts immune responses, parasite survival and pathogenesis of severe forms of malaria. We will focus on the role of complement receptor 1 and its associated polymorphisms in malaria immune complex clearance, as a mediator of parasite rosetting and as an entry receptor for P. falciparum invasion. Complement evasion strategies of P. falciparum parasites will also be highlighted. The sexual forms of the malaria parasites recruit the soluble human complement regulator Factor H to evade complement-mediated killing within the mosquito host. A novel evasion strategy is the deployment of parasite organelles to divert complement attack from infective blood stage parasites. Finally we outline the future challenge to understand the implications of these exploitation mechanisms in the interplay between successful infection of the host and pathogenesis observed in severe malaria. Copyright © 2015 Elsevier Ltd. All rights reserved.

Microbiological Quality of Fresh Nopal Juice

PubMed Central

Hernández-Anguiano, Ana María; Landa-Salgado, Patricia; Eslava-Campos, Carlos Alberto; Vargas-Hernández, Mateo; Patel, Jitendra

2016-01-01

The consumption of fresh nopal cactus juice is widely popular among health-conscious consumers in Mexico. The juice is prepared from fresh cladodes that have only been rinsed with tap water and are not subjected to a pasteurization or terminal bacterial reduction process. The aim of this study was to evaluate the microbial quality of commercially available fresh juices (n = 162) made with nopal in Texcoco, State of Mexico, during the summer and spring season. Standard microbiological methods, the PCR technique and the serological method were used for isolation and identification of bacteria. All samples contained total coliforms and 91% were positive for Escherichia coli. Although total coliforms and E. coli were detected throughout the study, their populations were significantly lower (p < 0.05) in winter and spring, respectively. Citrobacter youngae was found in 20% of the samples, an unidentified species of Citrobacter in 10%, C. freundii and Proteus mirabilis in 3%, and Salmonella Javiana in 1%. The presence of these microorganisms, especially Salmonella, in the nopal juices is unacceptable due to its health significance. The information generated in this study is relevant for human health risk assessment associated with the consumption of unpasteurized nopal juices and potential interventions to minimize pathogen contamination. PMID:27973398

Microbiological Quality of Fresh Nopal Juice.

PubMed

Hernández-Anguiano, Ana María; Landa-Salgado, Patricia; Eslava-Campos, Carlos Alberto; Vargas-Hernández, Mateo; Patel, Jitendra

2016-12-10

The consumption of fresh nopal cactus juice is widely popular among health-conscious consumers in Mexico. The juice is prepared from fresh cladodes that have only been rinsed with tap water and are not subjected to a pasteurization or terminal bacterial reduction process. The aim of this study was to evaluate the microbial quality of commercially available fresh juices ( n = 162) made with nopal in Texcoco, State of Mexico, during the summer and spring season. Standard microbiological methods, the PCR technique and the serological method were used for isolation and identification of bacteria. All samples contained total coliforms and 91% were positive for Escherichia coli . Although total coliforms and E. coli were detected throughout the study, their populations were significantly lower ( p < 0.05) in winter and spring, respectively. Citrobacter youngae was found in 20% of the samples, an unidentified species of Citrobacter in 10%, C. freundii and Proteus mirabilis in 3%, and Salmonella Javiana in 1%. The presence of these microorganisms, especially Salmonella , in the nopal juices is unacceptable due to its health significance. The information generated in this study is relevant for human health risk assessment associated with the consumption of unpasteurized nopal juices and potential interventions to minimize pathogen contamination.

Patterns of protective associations differ for antibodies to P. falciparum-infected erythrocytes and merozoites in immunity against malaria in children.

PubMed

Chan, Jo-Anne; Stanisic, Danielle I; Duffy, Michael F; Robinson, Leanne J; Lin, Enmoore; Kazura, James W; King, Christopher L; Siba, Peter M; Fowkes, Freya Ji; Mueller, Ivo; Beeson, James G

2017-12-01

Acquired antibodies play an important role in immunity to P. falciparum malaria and are typically directed towards surface antigens expressed by merozoites and infected erythrocytes (IEs). The importance of specific IE surface antigens as immune targets remains unclear. We evaluated antibodies and protective associations in two cohorts of children in Papua New Guinea. We used genetically-modified P. falciparum to evaluate the importance of PfEMP1 and a P. falciparum isolate with a virulent phenotype. Our findings suggested that PfEMP1 was the dominant target of antibodies to the IE surface, including functional antibodies that promoted opsonic phagocytosis by monocytes. Antibodies were associated with increasing age and concurrent parasitemia, and were higher among children exposed to a higher force-of-infection as determined using molecular detection. Antibodies to IE surface antigens were consistently associated with reduced risk of malaria in both younger and older children. However, protective associations for antibodies to merozoite surface antigens were only observed in older children. This suggests that antibodies to IE surface antigens, particularly PfEMP1, play an earlier role in acquired immunity to malaria, whereas greater exposure is required for protective antibodies to merozoite antigens. These findings have implications for vaccine design and serosurveillance of malaria transmission and immunity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prevalence of Plasmodium falciparum and non-P. falciparum infections in a highland district in Ghana, and the influence of HIV and sickle cell disease.

PubMed

Owusu, Ewurama D A; Brown, Charles A; Grobusch, Martin P; Mens, Petra

2017-04-24

In the past two decades, there has been a reported decline in malaria in Ghana and the rest of the world; yet it remains the number one cause of mortality and morbidity. Human immuno-deficiency virus (HIV) and sickle cell disease (SCD) share a common geographical space with malaria in sub-Saharan Africa and an interaction between these three conditions has been suggested. This study determined the Plasmodium falciparum and non-P. falciparum status of symptomatic and non-symptomatic residents of Mpraeso in the highlands of Kwahu-South district of Ghana based on evidence of current national decline. The influence of HIV and SCD on malaria was also determined. Participants were 354 symptomatic patients visiting the Kwahu Government Hospital and 360 asymptomatic residents of the district capital. This cross-sectional study was conducted during the minor rainy season (October-December 2014). Rapid diagnostic tests (RDT), blood film microscopy and real-time polymerase chain reaction assessment of blood were done. Participants who tested positive with RDT were treated with artemisinin-based combination therapy; and assessment of venous blood was repeated 7 days after treatment. HIV screening and haemoglobin genotyping was done. Univariate and multivariate regression analysis was used to determine the influence of SCD and HIV. Plasmodium falciparum was prevalent at 124/142 (87.3%). Plasmodium malariae was the only non-falciparum species detected at 18/142 (12.7%). HIV and SCD did not significantly increase odds of malaria infection. However, the use of ITN and recent anti-malarial intake significantly decreased the odds of being malaria infected by 0.45-fold and 0.46-fold respectively. Plasmodium falciparum and P. malariae infection are the prevailing species in the study area; albeit varying from the national average. HIV and SCD were not associated with the risk of having malaria.

Antiplasmodial activities of dyes against Plasmodium falciparum asexual and sexual stages: Contrasted uptakes of triarylmethanes Brilliant green, Green S (E142), and Patent Blue V (E131) by erythrocytes.

PubMed

Leba, Louis-Jérôme; Popovici, Jean; Estevez, Yannick; Pelleau, Stéphane; Legrand, Eric; Musset, Lise; Duplais, Christophe

2017-12-01

The search for safe antimalarial compounds acting against asexual symptom-responsible stages and sexual transmission-responsible forms of Plasmodium species is one of the major challenges in malaria elimination programs. So far, among current drugs approved for human use, only primaquine has transmission-blocking activity. The discovery of small molecules targeting different Plasmodium falciparum life stages remains a priority in antimalarial drug research. In this context, several independent studies have recently reported antiplasmodial and transmission-blocking activities of commonly used stains, dyes and fluorescent probes against P. falciparum including chloroquine-resistant isolates. Herein we have studied the antimalarial activities of dyes with different scaffold and we report that the triarylmethane dye (TRAM) Brilliant green inhibits the growth of asexual stages (IC 50  ≤ 2 μM) and has exflagellation-blocking activity (IC 50  ≤ 800 nM) against P. falciparum reference strains (3D7, 7G8) and chloroquine-resistant clinical isolate (Q206). In a second step we have investigated the antiplasmodial activities of two polysulfonated triarylmethane food dyes. Green S (E142) is weakly active against P. falciparum asexual stage (IC 50 ≃ 17 μM) whereas Patent Blue V (E131) is inactive in both antimalarial assays. By applying liquid chromatography techniques for the culture supernatant analysis after cell washings and lysis, we report the detection of Brilliant green in erythrocytes, the selective uptake of Green S (E142) by infected erythrocytes, whereas Patent Blue V (E131) could not be detected within non-infected and 3D7-infected erythrocytes. Overall, our results suggest that two polysulfonated food dyes might display different affinity with transporters or channels on infected RBC membrane. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Sequence analysis of DBL2β domain of vargene of Indonesian Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Sulistyaningsih, E.; Romadhon, B. D.; Palupi, I.; Hidayah, F.; Dewi, R.; Prasetyo, A.

2018-03-01

Malaria is a major health problem in tropical countries including Indonesia. The most deadly agent is Plasmodium falciparum. In P. falciparum infection, PfEMP1 is supposed to play an important role in the pathogenesis of malaria. PfEMP1 is encoded by var gene family, it is a polymorphic protein where the extra-cellular portion contains of three distinct binding domains: Duffy binding-like (DBL), Cysteine-rich interdomain regions (CIDR) and C2. PfEMP1 varies in domain composition and binding specificity. The study explored the characteristic of Indonesian DBL2β-var genes and investigated its role to the malaria outcome. Twenty blood samples from clinically mild to severe malaria patients in Jember, East Java were collected for DNA extraction. Diagnosis was confirmed by Giemsa-stained thick blood smear. PCR was conducted using specific primer targeting on the full-length of DBL2ß and resulted approximately single band of 1,7 kb in a sample. This band was observed only from severe malaria sample. Sequence analysis directly from PCR product showed 74-99% similarities with previous sequences in Gene Bank. In conclusion, the DBL2β domain of vargene of Indonesian isolates was 1603 nucleotides in length and there was a possible association of the existence of DBL2β domain with the severity of malaria outcome.

Fresh garlic: a possible vehicle for Salmonella Virchow.

PubMed

Bennett, C M; Dalton, C; Beers-Deeble, M; Milazzo, A; Kraa, E; Davos, D; Puech, M; Tan, A; Heuzenroeder, M W

2003-12-01

A sustained increase in Salmonella enterica serovar Virchow notifications in South Eastern Australia between September 1997 and May 1998 instigated a case-control study and environmental investigations. Cases were defined as having locally acquired culture-confirmed S. Virchow phage-type 8 infection and diarrhoeal disease. Matched controls were selected by progressive digit dialling based on cases' telephone numbers. An exposure and food history questionnaire was administered by telephone. Phage typing and pulse field gel electrophoresis were performed on case and environmental isolates. Thirty-two notifications of S. Virchow infection met the case definition, 37% reported bloody diarrhoea and S. Virchow was isolated from blood in 13% of cases. Twelve patients were admitted to hospital and one died. Fresh garlic (OR 4.1, 95% CI 1.3-12.8) and semi-dried tomatoes (OR 12.6, 95% CI 1.5-103.1) were associated with these cases. The associations remained significant after adjusting for sex and age. S. Virchow (PT 8) was cultured from two brands of semi-dried tomatoes associated with cases in two different states. We provide sufficient evidence for semi-dried tomatoes and fresh garlic to be considered as potential risk foods in future Salmonella outbreak investigations.

Importation of chloroquine-resistant Plasmodium falciparum by Guatemalan peacekeepers returning from the Democratic Republic of the Congo

PubMed Central

2013-01-01

Background Malaria elimination is being pursued in five of seven Central American countries. Military personnel returning from peacekeeping missions in sub-Saharan Africa could import chloroquine-resistant Plasmodium falciparum, posing a threat to elimination and to the continued efficacy of first-line chloroquine (CQ) treatment in these countries. This report describes the importation of P. falciparum from among 150 Guatemalan army special forces and support staff who spent ten months on a United Nations’ peacekeeping mission in the Democratic Republic of the Congo (DRC) in 2010. Methods Investigators reviewed patients’ medical charts and interviewed members of the contingent to identify malaria cases and risk factors for malaria acquisition. Clinical specimens were tested for malaria; isolated parasites were characterized molecularly for CQ resistance. Results Investigators identified 12 cases (8%) of laboratory-confirmed P. falciparum infection within the contingent; one case was from a soldier infected with a CQ-resistant pfcrt genotype resulting in his death. None of the contingent used an insecticide-treated bed net (ITN) or completely adhered to malaria chemoprophylaxis while in the DRC. Conclusion This report highlights the need to promote use of malaria prevention measures, in particular ITNs and chemoprophylaxis, among peacekeepers stationed in malaria-endemic areas. Countries attempting to eliminate malaria should consider appropriate methods to screen peacekeepers returning from endemic areas for malaria infections. Cases of malaria in travellers, immigrants and soldiers returning to Central America from countries with CQ-resistant malaria should be assumed to be carry resistant parasites and receive appropriate anti-malarial therapy to prevent severe disease and death. PMID:24060234

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.

ABO Blood Groups Influence Macrophage-mediated Phagocytosis of Plasmodium falciparum-infected Erythrocytes

PubMed Central

Branch, Donald R.; Hult, Annika K.; Olsson, Martin L.; Liles, W. Conrad; Cserti-Gazdewich, Christine M.; Kain, Kevin C.

2012-01-01

Erythrocyte polymorphisms associated with a survival advantage to Plasmodium falciparum infection have undergone positive selection. There is a predominance of blood group O in malaria-endemic regions, and several lines of evidence suggest that ABO blood groups may influence the outcome of P. falciparum infection. Based on the hypothesis that enhanced innate clearance of infected polymorphic erythrocytes is associated with protection from severe malaria, we investigated whether P. falciparum-infected O erythrocytes are more efficiently cleared by macrophages than infected A and B erythrocytes. We show that human macrophages in vitro and mouse monocytes in vivo phagocytose P. falciparum-infected O erythrocytes more avidly than infected A and B erythrocytes and that uptake is associated with increased hemichrome deposition and high molecular weight band 3 aggregates in infected O erythrocytes. Using infected A1, A2, and O erythrocytes, we demonstrate an inverse association of phagocytic capacity with the amount of A antigen on the surface of infected erythrocytes. Finally, we report that enzymatic conversion of B erythrocytes to type as O before infection significantly enhances their uptake by macrophages to observed level comparable to that with infected O wild-type erythrocytes. These data provide the first evidence that ABO blood group antigens influence macrophage clearance of P. falciparum-infected erythrocytes and suggest an additional mechanism by which blood group O may confer resistance to severe malaria. PMID:23071435

Antioxidant and Antiplasmodial Activities of Bergenin and 11-O-Galloylbergenin Isolated from Mallotus philippensis

PubMed Central

Khan, Hamayun; Amin, Hazrat; Ullah, Asad; Saba, Sumbal; Rafique, Jamal; Khan, Khalid; Ahmad, Nasir; Badshah, Syed Lal

2016-01-01

Two important biologically active compounds were isolated from Mallotus philippensis. The isolated compounds were characterized using spectroanalytical techniques and found to be bergenin (1) and 11-O-galloylbergenin (2). The in vitro antioxidant and antiplasmodial activities of the isolated compounds were determined. For the antioxidant potential, three standard analytical protocols, namely, DPPH radical scavenging activity (RSA), reducing power assay (RPA), and total antioxidant capacity (TAC) assay, were adopted. The results showed that compound 2 was found to be more potent antioxidant as compared to 1. Fascinatingly, compound 2 displayed better EC50 results as compared to α-tocopherol while being comparable with ascorbic acid. The antiplasmodial assay data showed that both the compound exhibited good activity against chloroquine sensitive strain of Plasmodium falciparum (D10) and IC50 values were found to be less than 8 μM. The in silico molecular docking analyses were also performed for the determination of binding affinity of the isolated compounds using P. falciparum proteins PfLDH and Pfg27. The results showed that compound 2 has high docking score and binding affinity to both protein receptors as compared to compound 1. The demonstrated biological potentials declared that compound 2 could be the better natural antioxidant and antiplasmodial candidate. PMID:26998192

Quantification of Histidine-Rich Protein 3 of Plasmodium falciparum.

PubMed

Palani, Balraj

2018-04-01

Malaria is a life-threatening infectious disease and continues to be a major public health crisis in many parts of the tropical world. Plasmodium falciparum is responsible for the majority of mortality and morbidity associated with malaria. During the intraerythrocytic cycle, P. falciparum releases three proteins with high histidine content as follows: histidine-rich protein 1 (HRP1), histidine-rich protein 2 (HRP2), and histidine-rich protein 3 (HRP3). Currently, most of the diagnostic tests of P. falciparum infection target HRP2, and a number of monoclonal antibodies (mAbs) against HRP2 have been developed for use in HRP2 detection and quantification. When parasites have HRP2 deletions, the detection of HRP3 could augment the sensitivity of the detection system. The combination of both HRP2 and HRP3 mAbs in the detection system will enhance the test sensitivity. In the HRP quantitative enzyme-linked immunosorbent assay (ELISA), both HRP2 and HRP3 contribute to the result, but the relative contribution of HRP2 and HRP3 was unable to investigate, because of the nonavailability of HRP3 specific antibody ELISA. Hence an ELISA test system based on HRP3 is also essential for detection and quantification. There is not much documented in the literature on HRP3 antigen and HRP3 specific mAbs and polyclonal antibodies (pAbs). In the present study, recombinant HRP3 was expressed in Escherichia coli and purified with Ni-NTA agarose column. The purified rHRP3 was used for the generation and characterization of monoclonal and pAbs. The purification of monoclonal and pAbs was done using a mixed-mode chromatography sorbent, phenylpropylamine HyperCel™. With the purified antibodies, a sandwich ELISA was developed. The sandwich ELISA method was explored to detect and quantify HRP3 of P. falciparum in the spent medium. The generated mAbs could be potentially used for the detection and quantification of P. falciparum HRP3.

Treatment of Chronic Asymptomatic Plasmodium falciparum Infection Does Not Increase the Risk of Clinical Malaria Upon Reinfection.

PubMed

Portugal, Silvia; Tran, Tuan M; Ongoiba, Aissata; Bathily, Aboudramane; Li, Shanping; Doumbo, Safiatou; Skinner, Jeff; Doumtabe, Didier; Kone, Younoussou; Sangala, Jules; Jain, Aarti; Davies, D Huw; Hung, Christopher; Liang, Li; Ricklefs, Stacy; Homann, Manijeh Vafa; Felgner, Philip L; Porcella, Stephen F; Färnert, Anna; Doumbo, Ogobara K; Kayentao, Kassoum; Greenwood, Brian M; Traore, Boubacar; Crompton, Peter D

2017-03-01

Chronic asymptomatic Plasmodium falciparum infections are common in endemic areas and are thought to contribute to the maintenance of malaria immunity. Whether treatment of these infections increases the subsequent risk of clinical episodes of malaria is unclear. In a 3-year study in Mali, asymptomatic individuals with or without P. falciparum infection at the end of the 6-month dry season were identified by polymerase chain reaction (PCR), and clinical malaria risk was compared during the ensuing 6-month malaria transmission season. At the end of the second dry season, 3 groups of asymptomatic children were identified: (1) children infected with P. falciparum as detected by rapid diagnostic testing (RDT) who were treated with antimalarials (n = 104), (2) RDT-negative children whose untreated P. falciparum infections were detected retrospectively by PCR (n = 55), and (3) uninfected children (RDT/PCR negative) (n = 434). Clinical malaria risk during 2 subsequent malaria seasons was compared. Plasmodium falciparum-specific antibody kinetics during the dry season were compared in children who did or did not harbor asymptomatic P. falciparum infections. Chronic asymptomatic P. falciparum infection predicted decreased clinical malaria risk during the subsequent malaria season(s); treatment of these infections did not alter this reduced risk. Plasmodium falciparum-specific antibodies declined similarly in children who did or did not harbor chronic asymptomatic P. falciparum infection during the dry season. These findings challenge the notion that chronic asymptomatic P. falciparum infection maintains malaria immunity and suggest that mass drug administration during the dry season should not increase the subsequent risk of clinical malaria. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Longitudinal study assessing the return of chloroquine susceptibility of Plasmodium falciparum in isolates from travellers returning from West and Central Africa, 2000–2011

PubMed Central

2013-01-01

Background Chloroquine (CQ) was the main malaria therapy worldwide from the 1940s until the 1990s. Following the emergence of CQ-resistant Plasmodium falciparum, most African countries discontinued the use of CQ, and now promote artemisinin-based combination therapy as the first-line treatment. This change was generally initiated during the last decade in West and Central Africa. The aim of this study is to describe the changes in CQ susceptibility in this African region, using travellers returning from this region as a sentinel system. Methods The study was conducted by the Malaria National Reference Centre, France. The database collated the pfcrtK76T molecular marker for CQ susceptibility and the in vitro response to CQ of parasites from travellers’ isolates returning from Senegal, Mali, Ivory Coast or Cameroon. As a proxy of drug pressure, data regarding CQ intake in febrile children were collated for the study period. Logistic regression models were used to detect trends in the proportions of CQ resistant isolates. Results A total of 2874 parasite isolates were genotyped between 2000–2011. The prevalence of the pfcrt76T mutant genotype significantly decreased for Senegal (from 78% to 47%), Ivory Coast (from 63% to 37%), Cameroon (from 90% to 59%) and remained stable for Mali. The geometric mean of the 50% inhibitory concentration (IC50) of CQ in vitro susceptibility and the proportion of resistant isolates (defining resistance as an IC50 value > 100 nM) significantly decreased for Senegal (from 86 nM (59%) to 39 nM (25%)), Mali (from 84 nM (50%) to 51 nM (31%)), Ivory Coast (from 75 nM (59%) to 29 nM (16%)) and Cameroon (from 181 nM (75%) to 51 nM (37%)). Both analyses (molecular and in vitro susceptibility) were performed for the 2004–2011 period, after the four countries had officially discontinued CQ and showed an accelerated decline of the resistant isolates for the four countries. Meanwhile, CQ use among children significantly deceased in this

Drug resistance associated genetic polymorphisms in Plasmodium falciparum and Plasmodium vivax collected in Honduras, Central America.

PubMed

Jovel, Irina T; Mejía, Rosa E; Banegas, Engels; Piedade, Rita; Alger, Jackeline; Fontecha, Gustavo; Ferreira, Pedro E; Veiga, Maria I; Enamorado, Irma G; Bjorkman, Anders; Ursing, Johan

2011-12-19

In Honduras, chloroquine and primaquine are recommended and still appear to be effective for treatment of Plasmodium falciparum and Plasmodium vivax malaria. The aim of this study was to determine the proportion of resistance associated genetic polymorphisms in P. falciparum and P. vivax collected in Honduras. Blood samples were collected from patients seeking medical attention at the Hospital Escuela in Tegucigalpa from 2004 to 2006 as well as three regional hospitals, two health centres and one regional laboratory during 2009. Single nucleotide polymorphisms in P. falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes and in P. vivax multidrug resistance 1 (pvmdr1) and dihydrofolate reductase (pvdhfr) genes were detected using PCR based methods. Thirty seven P. falciparum and 64 P. vivax samples were collected. All P. falciparum infections acquired in Honduras carried pfcrt, pfmdr1, pfdhps and pfdhfr alleles associated with chloroquine, amodiaquine and sulphadoxine-pyrimethamine sensitivity only. One patient with parasites acquired on a Pacific Island had pfcrt 76 T and pfmdr1 86Y alleles. That patient and a patient infected in West Africa had pfdhfr 51I, 59 R and 108 N alleles. Pvmdr1 976 F was found in 7/37 and two copies of pvmdr1 were found in 1/37 samples. Pvdhfr 57 L + 58 R was observed in 2/57 samples. The results indicate that P. falciparum from Honduras remain sensitive to chloroquine and sulphadoxine-pyrimethamine. This suggests that chloroquine and sulphadoxine-pyrimethamine should be efficacious for treatment of uncomplicated P. falciparum malaria, supporting current national treatment guidelines. However, genetic polymorphisms associated with chloroquine and sulphadoxine-pyrimethamine tolerance were detected in local P. vivax and imported P. falciparum infections. Continuous monitoring of the prevalence of drug resistant/tolerant P

Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum.

PubMed

Niemand, J; Louw, A I; Birkholtz, L; Kirk, K

2012-09-01

Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions, but does not cure infections in vivo. This may be due to replenishment of the parasite's intracellular polyamine pool via salvage of exogenous polyamines from the host. However, the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study, the uptake of the polyamines, putrescine and spermidine, into Plasmodium falciparum parasites functionally isolated from their host erythrocyte was investigated using radioisotope flux techniques. Both putrescine and spermidine were taken up into isolated parasites via a temperature-dependent process that showed cross-competition between different polyamines. There was also some inhibition of polyamine uptake by basic amino acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and spermidine taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated parasites was independent of extracellular Na(+) but increased with increasing external pH. Uptake also showed a marked dependence on the parasite's membrane potential, decreasing with membrane depolarization and increasing with membrane hyperpolarization. The data are consistent with polyamines being taken up into the parasite via an electrogenic uptake process, energised by the parasite's inwardly negative membrane potential. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Production of recombinant proteins from Plasmodium falciparum in Escherichia coli.

PubMed

Guerra, Ángela Patricia; Calvo, Eliana Patricia; Wasserman, Moisés; Chaparro-Olaya, Jacqueline

2016-02-23

The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum. However, the proteins of P. falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies.  To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8).  The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice.  The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli, compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies.  The use of genetically modified strains of E. coli was essential to achieve high expression levels of the four evaluated P. falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

Plasmodium falciparum, but not P. vivax, can induce erythrocytic apoptosis.

PubMed

Totino, Paulo Renato Rivas; Magalhães, Aline das Dores; Alves, Eliana Brasil; Costa, Monica Regina Farias; de Lacerda, Marcus Vinícius Guimarães; Daniel-Ribeiro, Cláudio Tadeu; Ferreira-da-Cruz, Maria de Fátima

2014-10-18

Apoptosis can occur in red blood cells (RBC) and seems to be involved in hematologic disorders related to many diseases. In malaria it is known that parasitized RBC (pRBC) is involved in the development of anemia and thrombosis; however, non-parasitized RBC (nRBC) apoptosis could amplify these malaria-associated hematologic events. In fact, in experimental malaria, increased levels of apoptosis were observed in nRBC during lethal Plasmodium yoelii 17XL infection, but in human malaria erythrocytic apoptosis has never been studied. The present study was performed to investigate if nRBC apoptosis also occurs in P. vivax and P. falciparum infections. Apoptosis of nRBC was evaluated in blood samples of P. vivax malaria patients and clinically healthly individuals living in Manaus, Brazil, both ex vivo and after incubation of RBC for 24 h. Additionally, the capacity of plasma from P. vivax or P. falciparum patients was tested for induction of in vitro apoptosis of normal RBC from a clinically healthy individual living in a non-endemic malaria region. Apoptosis was detected by flow cytometry using annexin V staining. In contrast to experimental malaria that significantly increased the levels of apoptotic nRBC both ex-vivo and after 24 h of incubation, no significant alteration on apoptotic nRBC rates was detected in P. vivax infected patients when compared with non-infected control individuals. Similar results were observed when plasma of these P. vivax patients was incubated with normal RBC. Conversely, plasma from P. falciparum-infected subjects induced significant apoptosis of these cells. Apoptosis of normal RBC can be induced by plasma from individuals with P. falciparum (but not with P. vivax) malaria. This finding could reflect the existence of erythrocytic apoptosis during infection that could contribute to the pathogenesis of hematological and vascular complications associated with falciparum malaria.

The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum.

PubMed

Bozdech, Zbynek; Llinás, Manuel; Pulliam, Brian Lee; Wong, Edith D; Zhu, Jingchun; DeRisi, Joseph L

2003-10-01

Plasmodium falciparum is the causative agent of the most burdensome form of human malaria, affecting 200-300 million individuals per year worldwide. The recently sequenced genome of P. falciparum revealed over 5,400 genes, of which 60% encode proteins of unknown function. Insights into the biochemical function and regulation of these genes will provide the foundation for future drug and vaccine development efforts toward eradication of this disease. By analyzing the complete asexual intraerythrocytic developmental cycle (IDC) transcriptome of the HB3 strain of P. falciparum, we demonstrate that at least 60% of the genome is transcriptionally active during this stage. Our data demonstrate that this parasite has evolved an extremely specialized mode of transcriptional regulation that produces a continuous cascade of gene expression, beginning with genes corresponding to general cellular processes, such as protein synthesis, and ending with Plasmodium-specific functionalities, such as genes involved in erythrocyte invasion. The data reveal that genes contiguous along the chromosomes are rarely coregulated, while transcription from the plastid genome is highly coregulated and likely polycistronic. Comparative genomic hybridization between HB3 and the reference genome strain (3D7) was used to distinguish between genes not expressed during the IDC and genes not detected because of possible sequence variations. Genomic differences between these strains were found almost exclusively in the highly antigenic subtelomeric regions of chromosomes. The simple cascade of gene regulation that directs the asexual development of P. falciparum is unprecedented in eukaryotic biology. The transcriptome of the IDC resembles a "just-in-time" manufacturing process whereby induction of any given gene occurs once per cycle and only at a time when it is required. These data provide to our knowledge the first comprehensive view of the timing of transcription throughout the intraerythrocytic

Trans-kingdom small RNA transfer during host-pathogen interactions: The case of P. falciparum and erythrocytes.

PubMed

Walzer, Katelyn A; Chi, Jen-Tsan

2017-04-03

This review focuses on the role of trans-kingdom movement of small RNA (sRNA) molecules between parasites, particularly Plasmodium falciparum, and their respective host cells. While the intercellular transfer of sRNAs within organisms is well recognized, recent studies illustrate many examples of trans-kingdom sRNA exchange within the context of host-parasite interactions. These interactions are predominantly found in the transfer of host sRNAs between erythrocytes and the invading P. falciparum, as well as other host cell types. In addition, parasite-encoded sRNAs can also be transferred to host cells to evade the immune system. The transport of these parasite sRNAs in the body fluids of the host may also offer means to detect and monitor the parasite infection. These isolated examples may only represent the tip of the iceberg in which the transfer of sRNA between host and parasites is a critical aspect of host-pathogen interactions. In addition, the levels of these sRNAs and their speed of transfer may vary dramatically under different contexts to push the biologic equilibrium toward the benefit of hosts vs. parasites. Therefore, these sRNA transfers may offer potential strategies to detect, prevent or treat parasite infections. Here, we review a brief history of the discovery of host erythrocyte sRNAs, their transfers and interactions in the context of P. falciparum infection. We also provide examples and discuss the functional significance of the reciprocal transfer of parasite-encoded sRNAs into hosts. These understandings of sRNA exchanges are put in the context of their implications for parasite pathogenesis, host defenses and the evolution of host polymorphisms driven by host interactions with these parasites.

P. falciparum infection and maternofetal antibody transfer in malaria-endemic settings of varying transmission

PubMed Central

Stanisic, Danielle; McGready, Rose; Chotivanich, Kesinee; Clapham, Caroline; Baiwog, Francesca; Pimanpanarak, Mupawjay; Siba, Peter; Mueller, Ivo; King, Christopher L.; Nosten, François; Beeson, James G.; Rogerson, Stephen; Simpson, Julie A.; Fowkes, Freya J. I.

2017-01-01

Introduction During pregnancy, immunoglobulin G (IgG) is transferred from the mother to the fetus, providing protection from disease in early infancy. Plasmodium falciparum infections may reduce maternofetal antibody transfer efficiency, but mechanisms remain unclear. Methods Mother-cord paired serum samples collected at delivery from Papua New Guinea (PNG) and the Thailand-Myanmar Border Area (TMBA) were tested for IgG1 and IgG3 to four P. falciparum antigens and measles antigen, as well as total serum IgG. Multivariable linear regression was conducted to assess the association of peripheral P. falciparum infection during pregnancy or placental P. falciparum infection assessed at delivery with maternofetal antibody transfer efficiency. Path analysis assessed the extent to which associations between P. falciparum infection and antibody transfer were mediated by gestational age at delivery or levels of maternal total serum IgG. Results Maternofetal antibody transfer efficiency of IgG1 and IgG3 was lower in PNG compared to TMBA (mean difference in cord antibody levels (controlling for maternal antibody levels) ranged from -0.88 to 0.09, median of -0.20 log2 units). Placental P. falciparum infections were associated with substantially lower maternofetal antibody transfer efficiency in PNG primigravid women (mean difference in cord antibody levels (controlling for maternal antibody levels) ranged from -0.62 to -0.10, median of -0.36 log2 units), but not multigravid women. The lower antibody transfer efficiency amongst primigravid women with placental infection was only partially mediated by gestational age at delivery (proportion indirect effect ranged from 0% to 18%), whereas no mediation effects of maternal total serum IgG were observed. Discussion Primigravid women may be at risk of impaired maternofetal antibody transport with placental P. falciparum infection. Direct effects of P. falciparum on the placenta, rather than earlier gestational age and elevated serum

P. falciparum infection and maternofetal antibody transfer in malaria-endemic settings of varying transmission.

PubMed

McLean, Alistair R D; Stanisic, Danielle; McGready, Rose; Chotivanich, Kesinee; Clapham, Caroline; Baiwog, Francesca; Pimanpanarak, Mupawjay; Siba, Peter; Mueller, Ivo; King, Christopher L; Nosten, François; Beeson, James G; Rogerson, Stephen; Simpson, Julie A; Fowkes, Freya J I

2017-01-01

During pregnancy, immunoglobulin G (IgG) is transferred from the mother to the fetus, providing protection from disease in early infancy. Plasmodium falciparum infections may reduce maternofetal antibody transfer efficiency, but mechanisms remain unclear. Mother-cord paired serum samples collected at delivery from Papua New Guinea (PNG) and the Thailand-Myanmar Border Area (TMBA) were tested for IgG1 and IgG3 to four P. falciparum antigens and measles antigen, as well as total serum IgG. Multivariable linear regression was conducted to assess the association of peripheral P. falciparum infection during pregnancy or placental P. falciparum infection assessed at delivery with maternofetal antibody transfer efficiency. Path analysis assessed the extent to which associations between P. falciparum infection and antibody transfer were mediated by gestational age at delivery or levels of maternal total serum IgG. Maternofetal antibody transfer efficiency of IgG1 and IgG3 was lower in PNG compared to TMBA (mean difference in cord antibody levels (controlling for maternal antibody levels) ranged from -0.88 to 0.09, median of -0.20 log2 units). Placental P. falciparum infections were associated with substantially lower maternofetal antibody transfer efficiency in PNG primigravid women (mean difference in cord antibody levels (controlling for maternal antibody levels) ranged from -0.62 to -0.10, median of -0.36 log2 units), but not multigravid women. The lower antibody transfer efficiency amongst primigravid women with placental infection was only partially mediated by gestational age at delivery (proportion indirect effect ranged from 0% to 18%), whereas no mediation effects of maternal total serum IgG were observed. Primigravid women may be at risk of impaired maternofetal antibody transport with placental P. falciparum infection. Direct effects of P. falciparum on the placenta, rather than earlier gestational age and elevated serum IgG, are likely responsible for the

Elucidation of sulfadoxine resistance with structural models of the bifunctional Plasmodium falciparum dihydropterin pyrophosphokinase-dihydropteroate synthase.

PubMed

de Beer, Tjaart A P; Louw, Abraham I; Joubert, Fourie

2006-07-01

Resistance of the most virulent human malaria parasite, Plasmodium falciparum, to antifolates is spreading with increasing speed, especially in Africa. Antifolate resistance is mainly caused by point mutations in the P. falciparum dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) target proteins. Homology models of the bifunctional P. falciparum dihydropterin pyrophosphokinase-dihydropteroate synthase (PPPK-DHPS) enzyme as well as the separate domains complete with bound substrates were constructed using the crystal structures of Saccharomyces cerevisiae (PPPK-DHPS), Mycobacterium tuberculosis (DHPS), Bacillus anthracis (DHPS), and Escherichia coli (PPPK) as templates. The resulting structures were subsequently solvated and refined using molecular dynamics. The active site residues of DHPS are highly conserved in S. cerevisiae, M. tuberculosis, E. coli, S. aureus, and B. anthracis, an attribute also shared by P. falciparum DHPS. Sulfadoxine was superimposed into the equivalent position of the p-aminobenzoic acid substrate and its binding parameters were refined using minimization and molecular dynamics. Sulfadoxine appears to interact mainly with P. falciparum DHPS mainly through hydrophobic interactions. Rational explanations are provided by the model for the sulfadoxine resistance-causing effects of four of the five known mutations in P. falciparum DHPS. A possible structure for the bifunctional PPPK-DHPS was derived from the structure from the S. cerevisiae bifunctional enzyme. The active site residues of P. falciparum PPPK are also conserved when compared to S. cerevisiae, Haemophilus influenzae, and E. coli. The informative nature of these models opens up avenues for structure-based drug design approaches toward the development of alternative and more effective inhibitors of P. falciparum PPPK-DHPS.

Complete Plasmodium falciparum liver-stage development in liver-chimeric mice.

PubMed

Vaughan, Ashley M; Mikolajczak, Sebastian A; Wilson, Elizabeth M; Grompe, Markus; Kaushansky, Alexis; Camargo, Nelly; Bial, John; Ploss, Alexander; Kappe, Stefan H I

2012-10-01

Plasmodium falciparum, which causes the most lethal form of human malaria, replicates in the host liver during the initial stage of infection. However, in vivo malaria liver-stage (LS) studies in humans are virtually impossible, and in vitro models of LS development do not reconstitute relevant parasite growth conditions. To overcome these obstacles, we have adopted a robust mouse model for the study of P. falciparum LS in vivo: the immunocompromised and fumarylacetoacetate hydrolase-deficient mouse (Fah-/-, Rag2-/-, Il2rg-/-, termed the FRG mouse) engrafted with human hepatocytes (FRG huHep). FRG huHep mice supported vigorous, quantifiable P. falciparum LS development that culminated in complete maturation of LS at approximately 7 days after infection, providing a relevant model for LS development in humans. The infections allowed observations of previously unknown expression of proteins in LS, including P. falciparum translocon of exported proteins 150 (PTEX150) and exported protein-2 (EXP-2), components of a known parasite protein export machinery. LS schizonts exhibited exoerythrocytic merozoite formation and merosome release. Furthermore, FRG mice backcrossed to the NOD background and repopulated with huHeps and human red blood cells supported reproducible transition from LS infection to blood-stage infection. Thus, these mice constitute reliable models to study human LS directly in vivo and demonstrate utility for studies of LS-to-blood-stage transition of a human malaria parasite.

[Construction of Plasmodium falciparum signal peptide peptidase-GFP mutant and its expression analysis in the malaria parasite].

PubMed

Li, Xue-rong; Wu, Yin-juan; Shang, Mei; Li, Ye; Xu, Jin; Yu, Xin-bing; Athar, Chishti

2014-08-01

To construct recombinant plasmid pSPPcGT which contains signal peptide peptidase gene of Plasmodium falciparum (PJSPP) and GFP, and transfect into P. falciparum (3D7 strain) to obtain mutant parasites which can express PJSPP-GFP. Plasmodium falciparum(3D7 strain) genomic DNA was extracted from cultured malaria parasites. The C-terminal region of PJSPP, an 883 bp gene fragment was amplified by PCR, and then cloned into pPM2GT vector to get recombinant vector pSPPcGT. The recombinant vectors were identified by PCR, double restriction enzyme digestion and DNA sequencing. pSPPcGT vector was transfected into malaria parasites. The positive clones were selected by adding inhibitor of Plasmodium falciparum dihydrofolate reductase WR99210 to the culture medium. The pSPP-GFP-transfected parasites were fixed with methanol, stained with DAPI, and observed under immunofluorescence microscope. The PJSPP-GFP expression in P. falciparum was identified by SDS-PAGE and Western blotting. The C-terminal region of PJSPP was amplified from P.falciparum (3D7 strain) genomic DNA by PCR with the length of 883 bp. The constructed recombinant vectors were identified by PCR screening, double restriction enzyme digestion and DNA sequencing. The pSPPcGT vector was transfected into P. falciparum and the positive clones were selected by WR99210. GFP fluorescence was observed in transfected parasites by immunofluorescence microscopy, and mainly located in the cytoplasm. The PJSPP-GFP expression in malaria parasites was confirmed by Western blotting with a relative molecular mass of Mr 64,000. Recombinant vector PJSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.

Selection of Plasmodium falciparum multidrug resistance gene 1 alleles in asexual stages and gametocytes by artemether-lumefantrine in Nigerian children with uncomplicated falciparum malaria.

PubMed

Happi, C T; Gbotosho, G O; Folarin, O A; Sowunmi, A; Hudson, T; O'Neil, M; Milhous, W; Wirth, D F; Oduola, A M J

2009-03-01

We assessed Plasmodium falciparum mdr1 (Pfmdr1) gene polymorphisms and copy numbers as well as P. falciparum Ca(2+) ATPase (PfATPase6) gene polymorphisms in 90 Nigerian children presenting with uncomplicated falciparum malaria and enrolled in a study of the efficacy of artemether-lumefantrine (AL). The nested PCR-restriction fragment length polymorphism and the quantitative real-time PCR methodologies were used to determine the alleles of the Pfmdr1 and PfATPase6 genes and the Pfmdr1 copy number variation, respectively, in patients samples collected prior to treatment and at the reoccurrence of parasites during a 42-day follow-up. The Pfmdr1 haplotype 86N-184F-1246D was significantly associated (P < 0.00001) with treatment failures and was selected for among posttreatment samples obtained from patients with newly acquired or recrudescing infections (P < 0.00001; chi(2) = 36.5) and in gametocytes (log rank statistic = 5; P = 0.0253) after treatment with AL. All pre- and posttreatment samples as well as gametocytes harbored a single copy of the Pfmdr1 gene and the wild-type allele (L89) at codon 89 of the PfATPase6 gene. These findings suggest that polymorphisms in the Pfmdr1 gene are under AL selection pressure. Pfmdr1 polymorphisms may result in reduction in the therapeutic efficacy of this newly adopted combination treatment for uncomplicated falciparum malaria in Saharan countries of Africa.

Direct detection of falciparum and non-falciparum malaria DNA from a drop of blood with high sensitivity by the dried-LAMP system.

PubMed

Hayashida, Kyoko; Kajino, Kiichi; Simukoko, Humphrey; Simuunza, Martin; Ndebe, Joseph; Chota, Amos; Namangala, Boniface; Sugimoto, Chihiro

2017-01-13

Because of the low sensitivity of conventional rapid diagnostic tests (RDTs) for malaria infections, the actual prevalence of the diseases, especially those caused by non-Plasmodium falciparum (non-Pf) species, in asymptomatic populations remain less defined in countries lacking in well-equipped facilities for accurate diagnoses. Our direct blood dry LAMP system (CZC-LAMP) was applied to the diagnosis of malaria as simple, rapid and highly sensitive method as an alternative for conventional RDTs in malaria endemic areas where laboratory resources are limited. LAMP primer sets for mitochondria DNAs of Plasmodium falciparum (Pf) and human-infective species other than Pf (non-Pf; P. vivax, P. ovale, P. malariae) were designed and tested by using human blood DNA samples from 74 residents from a malaria endemic area in eastern Zambia. These malaria dry-LAMPs were optimized for field or point-of-care operations, and evaluated in the field at a malaria endemic area in Zambia with 96 human blood samples. To determine the sensitivities and specificities, results obtained by the on-site LAMP diagnosis were compared with those by the nested PCR and nucleotide sequencing of its product. The dry LAMPs showed the sensitivities of 89.7% for Pf and 85.7% for non-Pf, and the specificities of 97.2% for Pf and 100% for non-Pf, with purified blood DNA samples. The direct blood LAMP diagnostic methods, in which 1 μl of anticoagulated blood were used as the template, showed the sensitivities of 98.1% for Pf, 92.1% for non-Pf, and the specificities of 98.1% for Pf, 100% for non-Pf. The prevalences of P. falciparum, P. malariae and P. ovale in the surveyed area were 52.4, 25.3 and 10.6%, respectively, indicating high prevalence of asymptomatic carriers in endemic areas in Zambia. We have developed new field-applicable malaria diagnostic tests. The malaria CZC-LAMPs showed high sensitivity and specificity to both P. falciparum and non-P. falciparum. These malaria CZC-LAMPs provide new

Factors affecting exflagellation of in vitro-cultivated Plasmodium falciparum gametocytes.

PubMed

Ogwan'g, R A; Mwangi, J K; Githure, J; Were, J B; Roberts, C R; Martin, S K

1993-07-01

The environment of Plasmodium falciparum gametocytes changes when they make the transition from the vertebrate to the invertebrate host. Gametocytes of this species cultivated in vitro were used to evaluate the effect of serum, pH, pCO2 tension, bicarbonate ion, and temperature on gamete formation. Temperature was the only factor responsible for keeping P. falciparum gametocytes in the inactivated state. Mature gametocytes held at temperatures above 30 degrees C remained quiescent in 10% serum, even at low ambient pCO2 tension, alkaline pH, and in the presence of 25 mM bicarbonate ion. When the temperature of the medium was allowed to drop below 30 degrees C, gametocytes emerged from the red blood cells and microgametocytes consistently exflagellated at pH 7.4, even in the absence of bicarbonate ion. With regard to bicarbonate ion, exflagellation in P. falciparum is similar to P. berghei and different from P. gallinaceum gametocytes, which have an obligate requirement for bicarbonate ion.

Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia.

PubMed

Lim, Pharath; Alker, Alisa P; Khim, Nimol; Shah, Naman K; Incardona, Sandra; Doung, Socheat; Yi, Poravuth; Bouth, Denis Mey; Bouchier, Christiane; Puijalon, Odile Mercereau; Meshnick, Steven R; Wongsrichanalai, Chansuda; Fandeur, Thierry; Le Bras, Jacques; Ringwald, Pascal; Ariey, Frédéric

2009-01-12

The combination of artesunate and mefloquine was introduced as the national first-line treatment for Plasmodium falciparum malaria in Cambodia in 2000. However, recent clinical trials performed at the Thai-Cambodian border have pointed to the declining efficacy of both artesunate-mefloquine and artemether-lumefantrine. Since pfmdr1 modulates susceptibility to mefloquine and artemisinin derivatives, the aim of this study was to assess the link between pfmdr1 copy number, in vitro susceptibility to individual drugs and treatment failure to combination therapy. Blood samples were collected from P. falciparum-infected patients enrolled in two in vivo efficacy studies in north-western Cambodia: 135 patients were treated with artemether-lumefantrine (AL group) in Sampovloun in 2002 and 2003, and 140 patients with artesunate-mefloquine (AM group) in Sampovloun and Veal Veng in 2003 and 2004. At enrollment, the in vitro IC50 was tested and the strains were genotyped for pfmdr1 copy number by real-time PCR. The pfmdr1 copy number was analysed for 115 isolates in the AM group, and for 109 isolates in the AL group. Parasites with increased pfmdr1 copy number had significantly reduced in vitro susceptibility to mefloquine, lumefantrine and artesunate. There was no association between pfmdr1 polymorphisms and in vitro susceptibilities. In the patients treated with AM, the mean pfmdr1copy number was lower in subjects with adequate clinical and parasitological response compared to those who experienced late treatment failure (n = 112, p < 0.001). This was not observed in the patients treated with AL (n = 96, p = 0.364). The presence of three or more copies of pfmdr1 were associated with recrudescence in artesunate-mefloquine treated patients (hazard ratio (HR) = 7.80 [95%CI: 2.09-29.10], N = 115), p = 0.002) but not with recrudescence in artemether-lumefantrine treated patients (HR = 1.03 [95%CI: 0.24-4.44], N = 109, p = 0.969). This study shows that pfmdr1 copy number is a

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.

Population genetics of Plasmodium falciparum and Plasmodium vivax and asymptomatic malaria in Temotu Province, Solomon Islands

PubMed Central

2013-01-01

Background Temotu Province, Solomon Islands is progressing toward malaria elimination. A baseline survey conducted in 2008 showed that most Plasmodium infections in the province were of low parasite density and asymptomatic infections. To better understand mechanisms underlying these malaria transmission characteristics genetic diversity and relationships among Plasmodium falciparum and Plasmodium vivax populations in the province were examined. Methods Forty-five P. falciparum and 67 P. vivax samples collected in the 2008 baseline survey were successfully genotyped using eight P. falciparum and seven P. vivax microsatellite markers. Genetic diversity, relationships and distribution of both P. falciparum and P. vivax populations were analysed. Results Plasmodium falciparum population exhibited low diversity with 19 haplotypes identified and had closely related clusters indicating clonal expansion. Interestingly, a dominant haplotype was significantly associated with fever and high parasite density. In contrast, the P. vivax population was highly diverse with 58 haplotypes identified that were not closely related. Parasite populations between different islands in the province showed low genetic differentiation. Conclusion The low diversity and clonal population of P. falciparum population may partially account for clinical immunity developed against illness. However, it is possible that importation of a new P. falciparum strain was the major cause of illness. High diversity in P. vivax population and low relatedness between strains suggested clinical immunity to P. vivax may be maintained by different mechanisms. The genetic diversity, population structure and distribution of strains indicate that transmission of P. falciparum was low, but that of P. vivax was still high in 2008. These data will be useful for assessing changes in malaria transmission resulting from interventions. PMID:24261646

Return of chloroquine sensitivity to Africa? Surveillance of African Plasmodium falciparum chloroquine resistance through malaria imported to China.

PubMed

Lu, Feng; Zhang, Meihua; Culleton, Richard L; Xu, Sui; Tang, Jianxia; Zhou, Huayun; Zhu, Guoding; Gu, Yaping; Zhang, Chao; Liu, Yaobao; Wang, Weiming; Cao, Yuanyuan; Li, Julin; He, Xinlong; Cao, Jun; Gao, Qi

2017-07-26

Chloroquine (CQ) was the cornerstone of anti-malarial treatment in Africa for almost 50 years, but has been widely withdrawn due to the emergence and spread of resistance. Recent reports have suggested that CQ-susceptibility may return following the cessation of CQ usage. Here, we monitor CQ sensitivity and determine the prevalence of genetic polymorphisms in the CQ resistance transporter gene (pfcrt) of Plasmodium falciparum isolates recently imported from Africa to China. Blood samples were collected from falciparum malaria patients returning to China from various countries in Africa. Isolates were tested for their sensitivity to CQ using the SYBR Green I test ex vivo, and for a subset of samples, in vitro following culture adaptation. Mutations at positions 72-76 and codon 220 of the pfcrt gene were analyzed by sequencing and confirmed by PCR-RFLP. Correlations between drug sensitivity and pfcrt polymorphisms were investigated. Of 32 culture adapted isolates assayed, 17 (53.1%), 6 (18.8%) and 9 (28.1%) were classified as sensitive, moderately resistant, and highly resistant, respectively. In vitro CQ susceptibility was related to point mutations in the pfcrt gene, the results indicating a strong association between pfcrt genotype and drug sensitivity. A total of 292 isolates were typed at the pfcrt locus, and the prevalence of the wild type (CQ sensitive) haplotype CVMNK in isolates from East, South, North, West and Central Africa were 91.4%, 80.0%, 73.3%, 53.3% and 51.7%, respectively. The only mutant haplotype observed was CVIET, and this was almost always linked to an additional mutation at A220S. Our results suggest that a reduction in drug pressure following withdrawal of CQ as a first-line drug may lead to a resurgence in CQ sensitive parasites. The prevalence of wild-type pfcrt CQ sensitive parasites from East, South and North Africa was higher than from the West and Central areas, but this varied greatly between countries. Further surveillance is

Constituents and secondary metabolite natural products in fresh and deteriorated cassava roots.

PubMed

Bayoumi, Soad A L; Rowan, Michael G; Beeching, John R; Blagbrough, Ian S

2010-04-01

A phytochemical analysis of cassava (Manihot esculenta Crantz) fresh roots and roots suffering from post-harvest physiological deterioration (PPD) has been carried out. The first isolation and identification of galactosyl diacylglycerides from fresh cassava roots is reported, as well as beta-carotene, linamarin, and beta-sitosterol glucopyranoside. The hydroxycoumarin scopoletin and its glucoside scopolin were identified from cassava roots during PPD, as well as trace quantities of esculetin and its glucoside esculin. There is no isoscopoletin in cassava roots during PPD. Copyright 2009 Elsevier Ltd. All rights reserved.

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

PubMed Central

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike; Ockenhouse, Christian F.; Emerling, Daniel; Glanville, Jacob; Volkmuth, Wayne; Flores-Garcia, Yevel; Zavala, Fidel; Ward, Andrew B.; King, C. Richter; Wilson, Ian A.

2017-01-01

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development in vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA)3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Together, these structural insights may aid in the design of a next-generation malaria vaccine. PMID:29138320

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

DOE PAGES

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike; ...

2017-11-14

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here in this paper, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development inmore » vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA) 3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Lastly, together, these structural insights may aid in the design of a next-generation malaria vaccine.« less

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here in this paper, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development inmore » vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA) 3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Lastly, together, these structural insights may aid in the design of a next-generation malaria vaccine.« less

Cultivation of Plasmodium falciparum parasites in a serum-free medium.

PubMed

Ofulla, A V; Okoye, V C; Khan, B; Githure, J I; Roberts, C R; Johnson, A J; Martin, S K

1993-09-01

The elimination of serum from Plasmodium falciparum culture media could decrease costs, enhance procurement, and improve the feasibility of large-scale production of parasite material. We provide a semi-defined, serum-free formulation, of commercially available constituents that supports P. falciparum parasite growth at rates comparable with those obtained with serum-supplemented media. The medium is composed of RPMI 1640 to which HEPES, extra glucose, bicarbonate, and hypoxanthine have been added. Bovine albumin and serum-derived, lipids-cholesterol-rich mixture are then used in place of serum.

Crystal Structure Analyses of the Fosmidomycin-Target Enzyme from Plasmodium Falciparum

NASA Astrophysics Data System (ADS)

Umeda, Tomonobu; Kusakabe, Yoshio; Tanaka, Nobutada

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. Fosmidomycin has proved to be efficient in the treatment of P. falciparum malaria through the inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an enzyme of the non-mevalonate pathway of isoprenoid biosynthesis, which is absent in humans. Crystal structure analyses of P. falciparum DXR (PfDXR) revealed that (i) an intrinsic flexibility of the PfDXR molecule accounts for the induced-fit movement to accommodate the bound inhibitor in the active site, and (ii) a cis arrangement of the oxygen atoms of the hydroxamate group of the bound inhibitor is essential for tight binding of the inhibitor to the active site metal. We believe that our study will serve as a useful guide to develop more potent PfDXR inhibitors.

Drug resistance associated genetic polymorphisms in Plasmodium falciparum and Plasmodium vivax collected in Honduras, Central America

PubMed Central

2011-01-01

Background In Honduras, chloroquine and primaquine are recommended and still appear to be effective for treatment of Plasmodium falciparum and Plasmodium vivax malaria. The aim of this study was to determine the proportion of resistance associated genetic polymorphisms in P. falciparum and P. vivax collected in Honduras. Methods Blood samples were collected from patients seeking medical attention at the Hospital Escuela in Tegucigalpa from 2004 to 2006 as well as three regional hospitals, two health centres and one regional laboratory during 2009. Single nucleotide polymorphisms in P. falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes and in P. vivax multidrug resistance 1 (pvmdr1) and dihydrofolate reductase (pvdhfr) genes were detected using PCR based methods. Results Thirty seven P. falciparum and 64 P. vivax samples were collected. All P. falciparum infections acquired in Honduras carried pfcrt, pfmdr1, pfdhps and pfdhfr alleles associated with chloroquine, amodiaquine and sulphadoxine-pyrimethamine sensitivity only. One patient with parasites acquired on a Pacific Island had pfcrt 76 T and pfmdr1 86Y alleles. That patient and a patient infected in West Africa had pfdhfr 51I, 59 R and 108 N alleles. Pvmdr1 976 F was found in 7/37 and two copies of pvmdr1 were found in 1/37 samples. Pvdhfr 57 L + 58 R was observed in 2/57 samples. Conclusion The results indicate that P. falciparum from Honduras remain sensitive to chloroquine and sulphadoxine-pyrimethamine. This suggests that chloroquine and sulphadoxine-pyrimethamine should be efficacious for treatment of uncomplicated P. falciparum malaria, supporting current national treatment guidelines. However, genetic polymorphisms associated with chloroquine and sulphadoxine-pyrimethamine tolerance were detected in local P. vivax and imported P. falciparum infections. Continuous monitoring of the prevalence

Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X.

PubMed

Li, Fengwu; Bounkeua, Viengngeun; Pettersen, Kenneth; Vinetz, Joseph M

2016-02-24

Plasmodium invasion of the mosquito midgut is a population bottleneck in the parasite lifecycle. Interference with molecular mechanisms by which the ookinete invades the mosquito midgut is one potential approach to developing malaria transmission-blocking strategies. Plasmodium aspartic proteases are one such class of potential targets: plasmepsin IV (known to be present in the asexual stage food vacuole) was previously shown to be involved in Plasmodium gallinaceum infection of the mosquito midgut, and plasmepsins VII and plasmepsin X (not known to be present in the asexual stage food vacuole) are upregulated in Plasmodium falciparum mosquito stages. These (and other) parasite-derived enzymes that play essential roles during ookinete midgut invasion are prime candidates for transmission-blocking vaccines. Reverse transcriptase PCR (RT-PCR) was used to determine timing of P. falciparum plasmepsin VII (PfPM VII) and plasmepsin X (PfPM X) mRNA transcripts in parasite mosquito midgut stages. Protein expression was confirmed by western immunoblot and immunofluorescence assays (IFA) using anti-peptide monoclonal antibodies (mAbs) against immunogenic regions of PfPM VII and PfPM X. These antibodies were also used in standard membrane feeding assays (SMFA) to determine whether inhibition of these proteases would affect parasite transmission to mosquitoes. The Mann-Whitney U test was used to analyse mosquito transmission assay results. RT-PCR, western immunoblot and immunofluorescence assay confirmed expression of PfPM VII and PfPM X in mosquito stages. Whereas PfPM VII was expressed in zygotes and ookinetes, PfPM X was expressed in gametes, zygotes, and ookinetes. Antibodies against PfPM VII and PfPM X decreased P. falciparum invasion of the mosquito midgut when used at high concentrations, indicating that these proteases play a role in Plasmodium mosquito midgut invasion. Failure to generate genetic knockouts of these genes limited determination of the precise role of

Plasmodium falciparum Malaria, Southern Algeria, 2007

PubMed Central

Gassen, Ibrahim; Khechache, Yacine; Lamali, Karima; Tchicha, Boualem; Brengues, Cécile; Menegon, Michela; Severini, Carlo; Fontenille, Didier; Harrat, Zoubir

2010-01-01

An outbreak of Plasmodium falciparum malaria occurred in Tinzaouatine in southern Algeria in 2007. The likely vector, Anopheles gambiae mosquitoes, had not been detected in Algeria. Genes for resistance to chloroquine were detected in the parasite. The outbreak shows the potential for an increase in malaria vectors in Algeria. PMID:20113565

Complete Plasmodium falciparum liver-stage development in liver-chimeric mice

PubMed Central

Vaughan, Ashley M.; Mikolajczak, Sebastian A.; Wilson, Elizabeth M.; Grompe, Markus; Kaushansky, Alexis; Camargo, Nelly; Bial, John; Ploss, Alexander; Kappe, Stefan H.I.

2012-01-01

Plasmodium falciparum, which causes the most lethal form of human malaria, replicates in the host liver during the initial stage of infection. However, in vivo malaria liver-stage (LS) studies in humans are virtually impossible, and in vitro models of LS development do not reconstitute relevant parasite growth conditions. To overcome these obstacles, we have adopted a robust mouse model for the study of P. falciparum LS in vivo: the immunocompromised and fumarylacetoacetate hydrolase–deficient mouse (Fah–/–, Rag2–/–, Il2rg–/–, termed the FRG mouse) engrafted with human hepatocytes (FRG huHep). FRG huHep mice supported vigorous, quantifiable P. falciparum LS development that culminated in complete maturation of LS at approximately 7 days after infection, providing a relevant model for LS development in humans. The infections allowed observations of previously unknown expression of proteins in LS, including P. falciparum translocon of exported proteins 150 (PTEX150) and exported protein-2 (EXP-2), components of a known parasite protein export machinery. LS schizonts exhibited exoerythrocytic merozoite formation and merosome release. Furthermore, FRG mice backcrossed to the NOD background and repopulated with huHeps and human red blood cells supported reproducible transition from LS infection to blood-stage infection. Thus, these mice constitute reliable models to study human LS directly in vivo and demonstrate utility for studies of LS–to–blood-stage transition of a human malaria parasite. PMID:22996664

Effect of a bacteriophage cocktail in combination with modified atmosphere packaging in controlling Listeria monocytogenes on fresh-cut spinach

USDA-ARS?s Scientific Manuscript database

A Listeria monocytogenes-specific bacteriophage cocktail (ListShield™) was evaluated for its activity against a nalidixic acid-resistant L. monocytogenes (Lm-NalR) isolate on fresh-cut spinach stored under modified atmosphere packaging (MAP) at various temperatures. Pieces (~2x2 cm2) of fresh spinac...

Evaluation of a rapid and inexpensive dipstick assay for the diagnosis of Plasmodium falciparum malaria.

PubMed Central

Mills, C. D.; Burgess, D. C.; Taylor, H. J.; Kain, K. C.

1999-01-01

Rapid, accurate and affordable methods are needed for the diagnosis of malaria. Reported here is an evaluation of a new immunochromatographic strip, the PATH Falciparum Malaria IC Strip, which is impregnated with an immobilized IgM monoclonal antibody that binds to the HRP-II antigen of Plasmodium falciparum. In contrast to other commercially available kits marketed for the rapid diagnosis of falciparum malaria, this kit should be affordable in the malaria-endemic world. Using microscopy and polymerase chain reaction (PCR)-based methods as reference standards, we compared two versions of the PATH test for the detection of P. falciparum infection in 200 febrile travellers. As determined by PCR and microscopy, 148 travellers had malaria, 50 of whom (33.8%) were infected with P. falciparum. Compared with PCR, the two versions of the PATH test had initial sensitivities of 90% and 88% and specificities of 97% and 96%, respectively, for the detection of falciparum malaria. When discrepant samples were retested blindly with a modified procedure (increased sample volume and longer washing step) the sensitivity and specificity of both kits improved to 96% and 99%, respectively. The two remaining false negatives occurred in samples with < 100 parasites per microliter of blood. The accuracy, simplicity and predicted low cost may make this test a useful diagnostic tool in malaria-endemic areas. PMID:10444878

[Evaluation of effect of prevention and control system for imported falciparum malaria in Hanjiang District].

PubMed

She, Guo-lin; Ma, Yu-Cai; Wang, Fu-biao

2013-08-01

To analyze the current situation of the comprehensive prevention and control system for imported falciparum malaria in Hanjiang District and evaluate its effect. According to the Management Scheme on Control of Imported Falciparum Malaria in Yangzhou City, the comprehensive prevention and control system for imported falciparum malaria was implemented, and the relevant malaria data were collected and analyzed statistically. The data included plasmodium blood test ratio of fever patients among exported labors and those returned, the ratio of laboratory-confirmed cases among all reported cases of falciparum malaria, the ratio of falciparum malaria patients who received the standard treatment within 24 hours after onset, etc from 2010 to 2012. After the implementation of the comprehensive prevention and control system, the confirmation ratio of falciparum malaria cases within 24 hours following first visit has reached 60.47%, the average time from first visit to confirmation has shortened to 1.8 d, and the average time from onset to confirmation has shortened to 3.7 d. The health education coverage ratio was 100%, the health knowledge awareness ratio was 95.56%, the ratio of patients seeking treatment on own initiative was 100%, the laboratory-confirmed ratio was 100%, and the ratio of standard treatment after malaria diagnosis was 100%. The comprehensive prevention and control system carried out by Hanjiang District has made remarkable achievements.

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

Evidence of non-Plasmodium falciparum malaria infection in Kédougou, Sénégal.

PubMed

Daniels, Rachel F; Deme, Awa Bineta; Gomis, Jules F; Dieye, Baba; Durfee, Katelyn; Thwing, Julie I; Fall, Fatou B; Ba, Mady; Ndiop, Medoune; Badiane, Aida S; Ndiaye, Yaye Die; Wirth, Dyann F; Volkman, Sarah K; Ndiaye, Daouda

2017-01-03

Expanded malaria control efforts in Sénégal have resulted in increased use of rapid diagnostic tests (RDT) to identify the primary disease-causing Plasmodium species, Plasmodium falciparum. However, the type of RDT utilized in Sénégal does not detect other malaria-causing species such as Plasmodium ovale spp., Plasmodium malariae, or Plasmodium vivax. Consequently, there is a lack of information about the frequency and types of malaria infections occurring in Sénégal. This study set out to better determine whether species other than P. falciparum were evident among patients evaluated for possible malaria infection in Kédougou, Sénégal. Real-time polymerase chain reaction speciation assays for P. vivax, P. ovale spp., and P. malariae were developed and validated by sequencing and DNA extracted from 475 Plasmodium falciparum-specific HRP2-based RDT collected between 2013 and 2014 from a facility-based sample of symptomatic patients from two health clinics in Kédougou, a hyper-endemic region in southeastern Sénégal, were analysed. Plasmodium malariae (n = 3) and P. ovale wallikeri (n = 2) were observed as co-infections with P. falciparum among patients with positive RDT results (n = 187), including one patient positive for all three species. Among 288 negative RDT samples, samples positive for P. falciparum (n = 24), P. ovale curtisi (n = 3), P. ovale wallikeri (n = 1), and P. malariae (n = 3) were identified, corresponding to a non-falciparum positivity rate of 2.5%. These findings emphasize the limitations of the RDT used for malaria diagnosis and demonstrate that non-P. falciparum malaria infections occur in Sénégal. Current RDT used for routine clinical diagnosis do not necessarily provide an accurate reflection of malaria transmission in Kédougou, Sénégal, and more sensitive and specific methods are required for diagnosis and patient care, as well as surveillance and elimination activities. These findings have implications for other

Allelic Diversity and Geographical Distribution of the Gene Encoding Plasmodium falciparum Merozoite Surface Protein-3 in Thailand.

PubMed

Sawaswong, Vorthon; Simpalipan, Phumin; Siripoon, Napaporn; Harnyuttanakorn, Pongchai; Pattaradilokrat, Sittiporn

2015-04-01

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines.

The weekly associations between climatic factors and Plasmodium vivax and Plasmodium falciparum malaria in China, 2005-2014.

PubMed

Hundessa, Samuel; Williams, Gail; Li, Shanshan; Guo, Jinpeng; Zhang, Wenyi; Guo, Yuming

2017-05-01

Meteorological factors play a crucial role in malaria transmission, but limited evidence is available from China. This study aimed to estimate the weekly associations between meteorological factors and Plasmodium vivax and Plasmodium falciparum malaria in China. The Distributed Lag Non-Linear Model was used to examine non-linearity and delayed effects of average temperature, rainfall, relative humidity, sunshine hours, wind speed and atmospheric pressure on malaria. Average temperature was associated with P. vivax and P. falciparum cases over long ranges of lags. The effect was more immediate on P. vivax (0-6 weeks) than on P. falciparum (1-9 weeks). Relative humidity was associated with P. vivax and P. falciparum over 8-10 weeks and 5-8 weeks lag, respectively. A significant effect of wind speed on P. vivax was observed at 0-2 weeks lag, but no association was found with P. falciparum. Rainfall had a decreasing effect on P. vivax, but no association was found with P. falciparum. Sunshine hours were negatively associated with P. falciparum, but the association was unclear for P. vixax. However, the effects of atmospheric pressure on both malaria types were not significant at any lag. Our study highlights a substantial effect of weekly climatic factors on P. vivax and P. falciparum malaria transmission in China, with different lags. This provides an evidence base for health authorities in developing a malaria early-warning system. © The Author 2017. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Selection of Plasmodium falciparum Multidrug Resistance Gene 1 Alleles in Asexual Stages and Gametocytes by Artemether-Lumefantrine in Nigerian Children with Uncomplicated Falciparum Malaria ▿

PubMed Central

Happi, C. T.; Gbotosho, G. O.; Folarin, O. A.; Sowunmi, A.; Hudson, T.; O'Neil, M.; Milhous, W.; Wirth, D. F.; Oduola, A. M. J.

2009-01-01

We assessed Plasmodium falciparum mdr1 (Pfmdr1) gene polymorphisms and copy numbers as well as P. falciparum Ca2+ ATPase (PfATPase6) gene polymorphisms in 90 Nigerian children presenting with uncomplicated falciparum malaria and enrolled in a study of the efficacy of artemether-lumefantrine (AL). The nested PCR-restriction fragment length polymorphism and the quantitative real-time PCR methodologies were used to determine the alleles of the Pfmdr1 and PfATPase6 genes and the Pfmdr1 copy number variation, respectively, in patients samples collected prior to treatment and at the reoccurrence of parasites during a 42-day follow-up. The Pfmdr1 haplotype 86N-184F-1246D was significantly associated (P < 0.00001) with treatment failures and was selected for among posttreatment samples obtained from patients with newly acquired or recrudescing infections (P < 0.00001; χ2 = 36.5) and in gametocytes (log rank statistic = 5; P = 0.0253) after treatment with AL. All pre- and posttreatment samples as well as gametocytes harbored a single copy of the Pfmdr1 gene and the wild-type allele (L89) at codon 89 of the PfATPase6 gene. These findings suggest that polymorphisms in the Pfmdr1 gene are under AL selection pressure. Pfmdr1 polymorphisms may result in reduction in the therapeutic efficacy of this newly adopted combination treatment for uncomplicated falciparum malaria in Saharan countries of Africa. PMID:19075074

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Acquired Antibodies to Merozoite Antigens in Children from Uganda with Uncomplicated or Severe Plasmodium falciparum Malaria

PubMed Central

Ahmed Ismail, Hodan; Ribacke, Ulf; Reiling, Linda; Normark, Johan; Egwang, Tom; Kironde, Fred; Beeson, James G.; Wahlgren, Mats

2013-01-01

Malaria can present itself as an uncomplicated or severe disease. We have here studied the quantity and quality of antibody responses against merozoite antigens, as well as multiplicity of infection (MOI), in children from Uganda. We found higher levels of IgG antibodies toward erythrocyte-binding antigen EBA181, MSP2 of Plasmodium falciparum 3D7 and FC27 (MSP2-3D7/FC27), and apical membrane antigen 1 (AMA1) in patients with uncomplicated malaria by enzyme-linked immunosorbent assay (ELISA) but no differences against EBA140, EBA175, MSP1, and reticulocyte-binding protein homologues Rh2 and Rh4 or for IgM against MSP2-3D7/FC27.Patients with uncomplicated malaria were also shown to have higher antibody affinities for AMA1 by surface plasmon resonance (SPR). Decreased invasion of two clinical P. falciparum isolates in the presence of patient plasma correlated with lower initial parasitemia in the patients, in contrast to comparisons of parasitemia to ELISA values or antibody affinities, which did not show any correlations. Analysis of the heterogeneity of the infections revealed a higher MOI in patients with uncomplicated disease, with the P. falciparum K1 MSP1 (MSP1-K1) and MSP2-3D7 being the most discriminative allelic markers. Higher MOIs also correlated positively with higher antibody levels in several of the ELISAs. In conclusion, certain antibody responses and MOIs were associated with differences between uncomplicated and severe malaria. When different assays were combined, some antibodies, like those against AMA1, seemed particularly discriminative. However, only decreased invasion correlated with initial parasitemia in the patient, signaling the importance of functional assays in understanding development of immunity against malaria and in evaluating vaccine candidates. PMID:23740926

Diversity of Antimicrobial Resistance and Virulence Determinants in Pseudomonas aeruginosa Associated with Fresh Vegetables

PubMed Central

Allydice-Francis, Kashina; Brown, Paul D.

2012-01-01

With the increased focus on healthy eating and consuming raw vegetables, this study assessed the extent of contamination of fresh vegetables by Pseudomonas aeruginosa in Jamaica and examined the antibiotic susceptibility profiles and the presence of various virulence associated determinants of P. aeruginosa. Analyses indicated that vegetables from retail markets and supermarkets were widely contaminated by P. aeruginosa; produce from markets were more frequently contaminated, but the difference was not significant. Lettuce and carrots were the most frequently contaminated vegetables, while tomatoes were the least. Pigment production (Pyoverdine, pyocyanin, pyomelanin and pyorubin), fluorescein and alginate were common in these isolates. Imipenem, gentamicin and ciprofloxacin were the most inhibitory antimicrobial agents. However, isolates were resistant or showed reduced susceptibility to ampicillin, chloramphenicol, sulphamethoxazole/trimethoprim and aztreonam, and up to 35% of the isolates were resistant to four antimicrobial agents. As many as 30% of the isolates were positive for the fpv1 gene, and 13% had multiple genes. Sixty-four percent of the isolates harboured an exoenzyme gene (exoS, exoT, exoU or exoY), and multiple exo genes were common. We conclude that P. aeruginosa is a major contaminant of fresh vegetables, which might be a source of infection for susceptible persons within the community. PMID:23213336

Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia.

PubMed

Mkulama, Mtawa A P; Chishimba, Sandra; Sikalima, Jay; Rouse, Petrica; Thuma, Philip E; Mharakurwa, Sungano

2008-05-21

In Zambia the first-line treatment for uncomplicated malaria is artemisinin combination therapy (ACT), with artemether-lumefantrine currently being used. However, the antifolate regimen, sulphadoxine-pyrimethamine (SP), remains the treatment of choice in children weighing less than 5 kg and also in expectant mothers. SP is also the choice drug for intermittent preventive therapy in pregnancy and serves as stand-by treatment during ACT stock outs. The current study assessed the status of Plasmodium falciparum point mutations associated with antifolate drug resistance in the area around Macha. A representative sample of 2,780 residents from the vicinity of Macha was screened for malaria by microscopy. At the same time, blood was collected onto filter paper and dried for subsequent P. falciparum DNA analysis. From 188 (6.8%) individuals that were thick film-positive, a simple random sub-set of 95 P. falciparum infections were genotyped for DHFR and DHPS antifolate resistance mutations, using nested PCR and allele-specific restriction enzyme digestion. Plasmodium falciparum field samples exhibited a high prevalence of antifolate resistance mutations, including the DHFR triple (Asn-108 + Arg-59 + Ile-51) mutant (41.3%) and DHPS double (Gly-437 + Glu-540) mutant (16%). The quintuple (DHFR triple + DHPS double) mutant was found in 4 (6.5%) of the samples. Levels of mutated parasites showed a dramatic escalation, relative to previous surveys since 1988. However, neither of the Val-16 and Thr-108 mutations, which jointly confer resistance to cycloguanil, was detectable among the human infections. The Leu-164 mutation, associated with high grade resistance to both pyrimethamine and cycloguanil, as a multiple mutant with Asn-108, Arg-59 and (or) Ile-51, was also absent. This study points to escalating levels of P. falciparum antifolate resistance in the vicinity of Macha. Continued monitoring is recommended to ensure timely policy revisions before widespread resistance exacts

Genetic Diversity of Plasmodium falciparum in Haiti: Insights from Microsatellite Markers

PubMed Central

Carter, Tamar E.; Malloy, Halley; Existe, Alexandre; Memnon, Gladys; St. Victor, Yves; Okech, Bernard A.; Mulligan, Connie J.

2015-01-01

Hispaniola, comprising Haiti and the Dominican Republic, has been identified as a candidate for malaria elimination. However, incomplete surveillance data in Haiti hamper efforts to assess the impact of ongoing malaria control interventions. Characteristics of the genetic diversity of Plasmodium falciparum populations can be used to assess parasite transmission, which is information vital to evaluating malaria elimination efforts. Here we characterize the genetic diversity of P. falciparum samples collected from patients at seven sites in Haiti using 12 microsatellite markers previously employed in population genetic analyses of global P. falciparum populations. We measured multiplicity of infections, level of genetic diversity, degree of population geographic substructure, and linkage disequilibrium (defined as non-random association of alleles from different loci). For low transmission populations like Haiti, we expect to see few multiple infections, low levels of genetic diversity, high degree of population structure, and high linkage disequilibrium. In Haiti, we found low levels of multiple infections (12.9%), moderate to high levels of genetic diversity (mean number of alleles per locus = 4.9, heterozygosity = 0.61), low levels of population structure (highest pairwise Fst = 0.09 and no clustering in principal components analysis), and moderate linkage disequilibrium (ISA = 0.05, P<0.0001). In addition, population bottleneck analysis revealed no evidence for a reduction in the P. falciparum population size in Haiti. We conclude that the high level of genetic diversity and lack of evidence for a population bottleneck may suggest that Haiti’s P. falciparum population has been stable and discuss the implications of our results for understanding the impact of malaria control interventions. We also discuss the relevance of parasite population history and other host and vector factors when assessing transmission intensity from genetic diversity data. PMID:26462203

Molecular surveillance for drug-resistant Plasmodium falciparum in clinical and subclinical populations from three border regions of Burma/Myanmar: cross-sectional data and a systematic review of resistance studies

PubMed Central

2012-01-01

Background Confirmation of artemisinin-delayed parasite clearance in Plasmodium falciparum along the Thai-Myanmar border has inspired a global response to contain and monitor drug resistance to avert the disastrous consequences of a potential spread to Africa. However, resistance data from Myanmar are sparse, particularly from high-risk areas where limited health services and decades of displacement create conditions for resistance to spread. Subclinical infections may represent an important reservoir for resistance genes that confer a fitness disadvantage relative to wild-type alleles. This study estimates the prevalence of resistance genotypes in three previously unstudied remote populations in Myanmar and tests the a priori hypothesis that resistance gene prevalence would be higher among isolates collected from subclinical infections than isolates collected from febrile clinical patients. A systematic review of resistance studies is provided for context. Methods Community health workers in Karen and Kachin States and an area spanning the Indo-Myanmar border collected dried blood spots from 988 febrile clinical patients and 4,591 villagers with subclinical infection participating in routine prevalence surveys. Samples positive for P. falciparum 18 s ribosomal RNA by real-time PCR were genotyped for P. falciparum multidrug resistance protein (pfmdr1) copy number and the pfcrt K76T polymorphism using multiplex real-time PCR. Results Pfmdr1 copy number increase and the pfcrt K76 polymorphism were determined for 173 and 269 isolates, respectively. Mean pfmdr1 copy number was 1.2 (range: 0.7 to 3.7). Pfmdr1 copy number increase was present in 17.5%, 9.6% and 11.1% of isolates from Karen and Kachin States and the Indo-Myanmar border, respectively. Pfmdr1 amplification was more prevalent in subclinical isolates (20.3%) than clinical isolates (6.4%, odds ratio 3.7, 95% confidence interval 1.1 - 12.5). Pfcrt K76T prevalence ranged from 90-100%. Conclusions Community

Molecular surveillance for drug-resistant Plasmodium falciparum in clinical and subclinical populations from three border regions of Burma/Myanmar: cross-sectional data and a systematic review of resistance studies.

PubMed

Brown, Tyler; Smith, Linda S; Oo, Eh Kalu Shwe; Shawng, Kum; Lee, Thomas J; Sullivan, David; Beyrer, Chris; Richards, Adam K

2012-09-19

Confirmation of artemisinin-delayed parasite clearance in Plasmodium falciparum along the Thai-Myanmar border has inspired a global response to contain and monitor drug resistance to avert the disastrous consequences of a potential spread to Africa. However, resistance data from Myanmar are sparse, particularly from high-risk areas where limited health services and decades of displacement create conditions for resistance to spread. Subclinical infections may represent an important reservoir for resistance genes that confer a fitness disadvantage relative to wild-type alleles. This study estimates the prevalence of resistance genotypes in three previously unstudied remote populations in Myanmar and tests the a priori hypothesis that resistance gene prevalence would be higher among isolates collected from subclinical infections than isolates collected from febrile clinical patients. A systematic review of resistance studies is provided for context. Community health workers in Karen and Kachin States and an area spanning the Indo-Myanmar border collected dried blood spots from 988 febrile clinical patients and 4,591 villagers with subclinical infection participating in routine prevalence surveys. Samples positive for P. falciparum 18 s ribosomal RNA by real-time PCR were genotyped for P. falciparum multidrug resistance protein (pfmdr1) copy number and the pfcrt K76T polymorphism using multiplex real-time PCR. Pfmdr1 copy number increase and the pfcrt K76 polymorphism were determined for 173 and 269 isolates, respectively. Mean pfmdr1 copy number was 1.2 (range: 0.7 to 3.7). Pfmdr1 copy number increase was present in 17.5%, 9.6% and 11.1% of isolates from Karen and Kachin States and the Indo-Myanmar border, respectively. Pfmdr1 amplification was more prevalent in subclinical isolates (20.3%) than clinical isolates (6.4%, odds ratio 3.7, 95% confidence interval 1.1 - 12.5). Pfcrt K76T prevalence ranged from 90-100%. Community health workers can contribute to molecular

Anemia Offers Stronger Protection Than Sickle Cell Trait Against the Erythrocytic Stage of Falciparum Malaria and This Protection Is Reversed by Iron Supplementation.

PubMed

Goheen, M M; Wegmüller, R; Bah, A; Darboe, B; Danso, E; Affara, M; Gardner, D; Patel, J C; Prentice, A M; Cerami, C

2016-12-01

Iron deficiency causes long-term adverse consequences for children and is the most common nutritional deficiency worldwide. Observational studies suggest that iron deficiency anemia protects against Plasmodium falciparum malaria and several intervention trials have indicated that iron supplementation increases malaria risk through unknown mechanism(s). This poses a major challenge for health policy. We investigated how anemia inhibits blood stage malaria infection and how iron supplementation abrogates this protection. This observational cohort study occurred in a malaria-endemic region where sickle-cell trait is also common. We studied fresh RBCs from anemic children (135 children; age 6-24months; hemoglobin <11g/dl) participating in an iron supplementation trial (ISRCTN registry, number ISRCTN07210906) in which they received iron (12mg/day) as part of a micronutrient powder for 84days. Children donated RBCs at baseline, Day 49, and Day 84 for use in flow cytometry-based in vitro growth and invasion assays with P. falciparum laboratory and field strains. In vitro parasite growth in subject RBCs was the primary endpoint. Anemia substantially reduced the invasion and growth of both laboratory and field strains of P. falciparum in vitro (~10% growth reduction per standard deviation shift in hemoglobin). The population level impact against erythrocytic stage malaria was 15.9% from anemia compared to 3.5% for sickle-cell trait. Parasite growth was 2.4 fold higher after 49days of iron supplementation relative to baseline (p<0.001), paralleling increases in erythropoiesis. These results confirm and quantify a plausible mechanism by which anemia protects African children against falciparum malaria, an effect that is substantially greater than the protection offered by sickle-cell trait. Iron supplementation completely reversed the observed protection and hence should be accompanied by malaria prophylaxis. Lower hemoglobin levels typically seen in populations of African

Genetic diversity and population structure of Plasmodium falciparum in Thailand, a low transmission country.

PubMed

Pumpaibool, Tepanata; Arnathau, Céline; Durand, Patrick; Kanchanakhan, Naowarat; Siripoon, Napaporn; Suegorn, Aree; Sitthi-Amorn, Chitr; Renaud, François; Harnyuttanakorn, Pongchai

2009-07-14

The population structure of the causative agents of human malaria, Plasmodium sp., including the most serious agent Plasmodium falciparum, depends on the local epidemiological and demographic situations, such as the incidence of infected people, the vector transmission intensity and migration of inhabitants (i.e. exchange between sites). Analysing the structure of P. falciparum populations at a large scale, such as continents, or with markers that are subject to non-neutral selection, can lead to a masking and misunderstanding of the effective process of transmission. Thus, knowledge of the genetic structure and organization of P. falciparum populations in a particular area with neutral genetic markers is needed to understand which epidemiological factors should be targeted for disease control. Limited reports are available on the population genetic diversity and structure of P. falciparum in Thailand, and this is of particular concern at the Thai-Myanmar and Thai-Cambodian borders, where there is a reported high resistance to anti-malarial drugs, for example mefloquine, with little understanding of its potential gene flow. The diversity and genetic differentiation of P. falciparum populations were analysed using 12 polymorphic apparently neutral microsatellite loci distributed on eight of the 14 different chromosomes. Samples were collected from seven provinces in the western, eastern and southern parts of Thailand. A strong difference in the nuclear genetic structure was observed between most of the assayed populations. The genetic diversity was comparable to the intermediate level observed in low P. falciparum transmission areas (average HS = 0.65 +/- 0.17), where the lowest is observed in South America and the highest in Africa. However, uniquely the Yala province, had only a single multilocus genotype present in all samples, leading to a strong geographic differentiation when compared to the other Thai populations during this study. Comparison of the genetic

Towards development of aptamers that specifically bind to lactate dehydrogenase of Plasmodium falciparum through epitopic targeting.

PubMed

Frith, Kelly-Anne; Fogel, Ronen; Goldring, J P Dean; Krause, Robert G E; Khati, Makobetsa; Hoppe, Heinrich; Cromhout, Mary E; Jiwaji, Meesbah; Limson, Janice L

2018-05-03

Early detection is crucial for the effective treatment of malaria, particularly in those cases infected with Plasmodium falciparum. There is a need for diagnostic devices with the capacity to distinguish P. falciparum from other strains of malaria. Here, aptamers generated against targeted species-specific epitopes of P. falciparum lactate dehydrogenase (rPfLDH) are described. Two classes of aptamers bearing high binding affinity and specificity for recombinant P. falciparum lactate dehydrogenase (rPfLDH) and P. falciparum-specific lactate dehydrogenase epitopic oligopeptide (LDHp) were separately generated. Structurally-relevant moieties with particular consensus sequences (GGTAG and GGCG) were found in aptamers reported here and previously published, confirming their importance in recognition of the target, while novel moieties particular to this work (ATTAT and poly-A stretches) were identified. Aptamers with diagnostically-supportive functions were synthesized, prime examples of which are the aptamers designated as LDHp 1, LDHp 11 and rLDH 4 and rLDH 15 in work presented herein. Of the sampled aptamers raised against the recombinant protein, rLDH 4 showed the highest binding to the target rPfLDH in the ELONA assay, with both rLDH 4 and rLDH 15 indicating an ability to discriminate between rPfLDH and rPvLDH. LDHp 11 was generated against a peptide selected as a unique P. falciparum LDH peptide. The aptamer, LDHp 11, like antibodies against the same peptide, only detected rPfLDH and discriminated between rPfLDH and rPvLDH. This was supported by affinity binding experiments where only aptamers generated against a unique species-specific epitope showed an ability to preferentially bind to rPfLDH relative to rPvLDH rather than those generated against the whole recombinant protein. In addition, rLDH 4 and LDHp 11 demonstrated in situ binding to P. falciparum cells during confocal microscopy. The utilization and application of LDHp 11, an aptamer generated against a

Resisting and tolerating P. falciparum in pregnancy under different malaria transmission intensities.

PubMed

Ndam, Nicaise Tuikue; Mbuba, Emmanuel; González, Raquel; Cisteró, Pau; Kariuki, Simon; Sevene, Esperança; Rupérez, María; Fonseca, Ana Maria; Vala, Anifa; Maculuve, Sonia; Jiménez, Alfons; Quintó, Llorenç; Ouma, Peter; Ramharter, Michael; Aponte, John J; Nhacolo, Arsenio; Massougbodji, Achille; Briand, Valerie; Kremsner, Peter G; Mombo-Ngoma, Ghyslain; Desai, Meghna; Macete, Eusebio; Cot, Michel; Menéndez, Clara; Mayor, Alfredo

2017-07-17

Resistance and tolerance to Plasmodium falciparum can determine the progression of malaria disease. However, quantitative evidence of tolerance is still limited. We investigated variations in the adverse impact of P. falciparum infections among African pregnant women under different intensities of malaria transmission. P. falciparum at delivery was assessed by microscopy, quantitative PCR (qPCR) and placental histology in 946 HIV-uninfected and 768 HIV-infected pregnant women from Benin, Gabon, Kenya and Mozambique. Resistance was defined by the proportion of submicroscopic infections and the levels of anti-parasite antibodies quantified by Luminex, and tolerance by the relationship of pregnancy outcomes with parasite densities at delivery. P. falciparum prevalence by qPCR in peripheral and/or placental blood of HIV-uninfected Mozambican, Gabonese and Beninese women at delivery was 6% (21/340), 11% (28/257) and 41% (143/349), respectively. The proportion of peripheral submicroscopic infections was higher in Benin (83%) than in Mozambique (60%) and Gabon (55%; P = 0.033). Past or chronic placental P. falciparum infection was associated with an increased risk of preterm birth in Mozambican newborns (OR = 7.05, 95% CI 1.79 to 27.82). Microscopic infections were associated with reductions in haemoglobin levels at delivery among Mozambican women (-1.17 g/dL, 95% CI -2.09 to -0.24) as well as with larger drops in haemoglobin levels from recruitment to delivery in Mozambican (-1.66 g/dL, 95% CI -2.68 to -0.64) and Gabonese (-0.91 g/dL, 95% CI -1.79 to -0.02) women. Doubling qPCR-peripheral parasite densities in Mozambican women were associated with decreases in haemoglobin levels at delivery (-0.16 g/dL, 95% CI -0.29 to -0.02) and increases in the drop of haemoglobin levels (-0.29 g/dL, 95% CI -0.44 to -0.14). Beninese women had higher anti-parasite IgGs than Mozambican women (P < 0.001). No difference was found in the proportion of submicroscopic

Risk factors of shock in severe falciparum malaria.

PubMed

Arnold, Brendan J; Tangpukdee, Noppadon; Krudsood, Srivicha; Wilairatana, Polrat

2013-07-04

The objective of this study was to determine the risk factors for the development of shock in adult patients admitted with severe falciparum malaria. As an unmatched case-control study, the records of patients who were admitted to the Bangkok Hospital for Tropical Diseases, Thailand, between the years 2000-2010, were reviewed. One hundred patients with severe falciparum malaria and shock, and another 100 patients with severe malaria but without shock were studied. Demographics, presenting symptoms, physical observations, and laboratory data of these patients were analyzed. Five risk factors for the development of shock were identified: female gender (OR 6.16; 95% CI 3.17-11.97), red cell distribution width (RDW) >15% (adjusted OR 2.90; 95% CI 1.11-7.57), anorexia (adjusted OR 2.76; 95% CI 1.03-7.39), hypoalbuminemia (adjusted OR 2.19; 95% CI 1.10-4.34), and BUN-creatinine ratio >20 (adjusted OR 2.38; 95% CI 1.22-4.64). Diarrhea was found to be a protective factor (adjusted OR 0.33; 95% CI 0.14-0.78). Metabolic acidosis was only weakly correlated to mean arterial blood pressure on admission (r(s) = 0.23). Female gender was the strongest risk factor for the development of shock. We concluded that female gender, RDW >15%, anorexia, hypoalbuminemia, and BUN-creatinine ratio >20 were risk factors of shock development in severe falciparum malaria.

Two novel aromatic glucosides, marylaurencinosides D and E, from the fresh flowers of Cymbidium Great Flower 'Marylaurencin'.

PubMed

Yoshikawa, Kazuko; Okahuji, Mariko; Iseki, Kanako; Ito, Takuya; Asakawa, Yoshinori; Kawano, Sachiko; Hashimoto, Toshihiro

2014-04-01

Two novel aromatic glucosides, named marylaurencinosides D (1) and E (2), were isolated from the fresh flowers of Cymbidium Great Flower 'Marylaurencin'. In addition, eight known aromatic compounds (3-10) were isolated. These structures were determined on the basis of NMR experiments as well as chemical evidence.

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

Host age and Plasmodium falciparum multiclonality are associated with gametocyte prevalence: a 1-year prospective cohort study.

PubMed

Adomako-Ankomah, Yaw; Chenoweth, Matthew S; Tocker, Aaron M; Doumbia, Saibou; Konate, Drissa; Doumbouya, Mory; Keita, Abdoul S; Anderson, Jennifer M; Fairhurst, Rick M; Diakite, Mahamadou; Miura, Kazutoyo; Long, Carole A

2017-11-21

Since Plasmodium falciparum transmission relies exclusively on sexual-stage parasites, several malaria control strategies aim to disrupt this step of the life cycle. Thus, a better understanding of which individuals constitute the primary gametocyte reservoir within an endemic population, and the temporal dynamics of gametocyte carriage, especially in seasonal transmission settings, will not only support the effective implementation of current transmission control programmes, but also inform the design of more targeted strategies. A 1-year prospective cohort study was initiated in June 2013 with the goal of assessing the longitudinal dynamics of P. falciparum gametocyte carriage in a village in Mali with intense seasonal malaria transmission. A cohort of 500 individuals aged 1-65 years was recruited for this study. Gametocyte prevalence was measured monthly using Pfs25-specific RT-PCR, and analysed for the effects of host age and gender, seasonality, and multiclonality of P. falciparum infection over 1 year. Most P. falciparum infections (51-89%) in this population were accompanied by gametocytaemia throughout the 1-year period. Gametocyte prevalence among P. falciparum-positive individuals (proportion of gametocyte positive infections) was associated with age (p = 0.003) but not with seasonality (wet vs. dry) or gender. The proportion of gametocyte positive infections were similarly high in children aged 1-17 years (74-82% on median among 5 age groups), while older individuals had relatively lower proportion, and those aged > 35 years (median of 43%) had significantly lower than those aged 1-17 years (p < 0.05). Plasmodium falciparum-positive individuals with gametocytaemia were found to have significantly higher P. falciparum multiclonality than those without gametocytaemia (p < 0.033 in two different analyses). Taken together, these results suggest that a substantial proportion of Pf-positive individuals carries gametocytes throughout the year, and

Allelic Diversity and Geographical Distribution of the Gene Encoding Plasmodium falciparum Merozoite Surface Protein-3 in Thailand

PubMed Central

Sawaswong, Vorthon; Simpalipan, Phumin; Siripoon, Napaporn; Harnyuttanakorn, Pongchai; Pattaradilokrat, Sittiporn

2015-01-01

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines. PMID:25925176

[Detection of Plasmodium falciparum by using magnetic nanoparticles separation-based quantitative real-time PCR assay].

PubMed

Wang, Fei; Tian, Yin; Yang, Jing; Sun, Fu-Jun; Sun, Ning; Liu, Bi-Yong; Tian, Rui; Ge, Guang-Lu; Zou, Ming-qiang; Deng, Cong-liang; Liu, Yi

2014-10-01

To establish a magnetic nanoparticles separation-based quantitative real-time PCR (RT-PCR) assay for fast and accurate detection of Plasmodium falciparum and providing a technical support for improving the control and prevention of imported malaria. According to the conserved sequences of the P. falciparum genome 18SrRNA, the species-specific primers and probe were designed and synthetized. The RT-PCR was established by constructing the plasmid standard, fitting the standard curve and using magnetic nanoparticles separation. The sensitivity and specificity of the assay were evaluated. The relationship between the threshold cycle (Ct) and logarithm of initial templates copies was linear over a range of 2.5 x 10(1) to 2.5 x 10(8) copies/μl (R2 = 0.999). Among 13 subjects of entry frontier, a P. falciparum carrier with low load was detected by using the assay and none was detected with the conventional examinations (microscopic examinations and rapid tests). This assay shows a high sensitivity in detection of P. falciparum, with rapid and accurate characteristics, and is especially useful in diagnosis of P. falciparum infectors with low parasitaemia at entry-exit frontier ports.

Spread of artemisinin resistance in Plasmodium falciparum malaria.

PubMed

Ashley, Elizabeth A; Dhorda, Mehul; Fairhurst, Rick M; Amaratunga, Chanaki; Lim, Parath; Suon, Seila; Sreng, Sokunthea; Anderson, Jennifer M; Mao, Sivanna; Sam, Baramey; Sopha, Chantha; Chuor, Char Meng; Nguon, Chea; Sovannaroth, Siv; Pukrittayakamee, Sasithon; Jittamala, Podjanee; Chotivanich, Kesinee; Chutasmit, Kitipumi; Suchatsoonthorn, Chaiyaporn; Runcharoen, Ratchadaporn; Hien, Tran Tinh; Thuy-Nhien, Nguyen Thanh; Thanh, Ngo Viet; Phu, Nguyen Hoan; Htut, Ye; Han, Kay-Thwe; Aye, Kyin Hla; Mokuolu, Olugbenga A; Olaosebikan, Rasaq R; Folaranmi, Olaleke O; Mayxay, Mayfong; Khanthavong, Maniphone; Hongvanthong, Bouasy; Newton, Paul N; Onyamboko, Marie A; Fanello, Caterina I; Tshefu, Antoinette K; Mishra, Neelima; Valecha, Neena; Phyo, Aung Pyae; Nosten, Francois; Yi, Poravuth; Tripura, Rupam; Borrmann, Steffen; Bashraheil, Mahfudh; Peshu, Judy; Faiz, M Abul; Ghose, Aniruddha; Hossain, M Amir; Samad, Rasheda; Rahman, M Ridwanur; Hasan, M Mahtabuddin; Islam, Akhterul; Miotto, Olivo; Amato, Roberto; MacInnis, Bronwyn; Stalker, Jim; Kwiatkowski, Dominic P; Bozdech, Zbynek; Jeeyapant, Atthanee; Cheah, Phaik Yeong; Sakulthaew, Tharisara; Chalk, Jeremy; Intharabut, Benjamas; Silamut, Kamolrat; Lee, Sue J; Vihokhern, Benchawan; Kunasol, Chanon; Imwong, Mallika; Tarning, Joel; Taylor, Walter J; Yeung, Shunmay; Woodrow, Charles J; Flegg, Jennifer A; Das, Debashish; Smith, Jeffery; Venkatesan, Meera; Plowe, Christopher V; Stepniewska, Kasia; Guerin, Philippe J; Dondorp, Arjen M; Day, Nicholas P; White, Nicholas J

2014-07-31

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. 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. 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 infections (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. 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 International Development and others; Clinical

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

Biomarkers of Plasmodium falciparum Infection during Pregnancy in Women Living in Northeastern Tanzania

PubMed Central

Boström, Stéphanie; Ibitokou, Samad; Oesterholt, Mayke; Schmiegelow, Christentze; Persson, Jan-Olov; Minja, Daniel; Lusingu, John; Lemnge, Martha; Fievet, Nadine; Deloron, Philippe; Luty, Adrian J. F.; Troye-Blomberg, Marita

2012-01-01

In pregnant women, Plasmodium falciparum infections are an important cause of maternal morbidity as well as fetal and neonatal mortality. Erythrocytes infected by these malaria-causing parasites accumulate through adhesive interactions in placental intervillous spaces, thus evading detection in peripheral blood smears. Sequestered infected erythrocytes induce inflammation, offering the possibility of detecting inflammatory mediators in peripheral blood that could act as biomarkers of placental infection. In a longitudinal, prospective study in Tanzania, we quantified a range of different cytokines, chemokines and angiogenic factors in peripheral plasma samples, taken on multiple sequential occasions during pregnancy up to and including delivery, from P. falciparum-infected women and matched uninfected controls. The results show that during healthy, uninfected pregnancies the levels of most of the panel of molecules we measured were largely unchanged except at delivery. In women with P. falciparum, however, both comparative and longitudinal assessments consistently showed that the levels of IL-10 and IP-10 increased significantly whilst that of RANTES decreased significantly, regardless of gestational age at the time the infection was detected. ROC curve analysis indicated that a combination of increased IL-10 and IP-10 levels and decreased RANTES levels might be predictive of P. falciparum infections. In conclusion, our data suggest that host biomarkers in peripheral blood may represent useful diagnostic markers of P. falciparum infection during pregnancy, but placental histology results would need to be included to verify these findings. PMID:23155405

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

A Plasmodium falciparum strain expressing GFP throughout the parasite's life-cycle.

PubMed

Talman, Arthur M; Blagborough, Andrew M; Sinden, Robert E

2010-02-10

The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP) throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito.

Striving for safety in fresh and fresh-cut fruits and vegetables

USDA-ARS?s Scientific Manuscript database

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 ...

Rapid diagnostic tests for diagnosing uncomplicated non-falciparum or Plasmodium vivax malaria in endemic countries.

PubMed

Abba, Katharine; Kirkham, Amanda J; Olliaro, Piero L; Deeks, Jonathan J; Donegan, Sarah; Garner, Paul; Takwoingi, Yemisi

2014-12-18

In settings where both Plasmodium vivax and Plasmodium falciparum infection cause malaria, rapid diagnostic tests (RDTs) need to distinguish which species is causing the patients' symptoms, as different treatments are required. Older RDTs incorporated two test lines to distinguish malaria due to P. falciparum, from malaria due to any other Plasmodium species (non-falciparum). These RDTs can be classified according to which antibodies they use: Type 2 RDTs use HRP-2 (for P. falciparum) and aldolase (all species); Type 3 RDTs use HRP-2 (for P. falciparum) and pLDH (all species); Type 4 use pLDH (fromP. falciparum) and pLDH (all species).More recently, RDTs have been developed to distinguish P. vivax parasitaemia by utilizing a pLDH antibody specific to P. vivax. To assess the diagnostic accuracy of RDTs for detecting non-falciparum or P. vivax parasitaemia in people living in malaria-endemic areas who present to ambulatory healthcare facilities with symptoms suggestive of malaria, and to identify which types and brands of commercial test best detect non-falciparum and P. vivax malaria. We undertook a comprehensive search of the following databases up to 31 December 2013: Cochrane Infectious Diseases Group Specialized Register; MEDLINE; EMBASE; MEDION; Science Citation Index; Web of Knowledge; African Index Medicus; LILACS; and IndMED. Studies comparing RDTs with a reference standard (microscopy or polymerase chain reaction) in blood samples from a random or consecutive series of patients attending ambulatory health facilities with symptoms suggestive of malaria in non-falciparum endemic areas. For each study, two review authors independently extracted a standard set of data using a tailored data extraction form. We grouped comparisons by type of RDT (defined by the combinations of antibodies used), and combined in meta-analysis where appropriate. Average sensitivities and specificities are presented alongside 95% confidence intervals (95% CI). We included 47 studies

Relative Susceptibilities of ABO Blood Groups to Plasmodium falciparum Malaria in Ghana.

PubMed

Afoakwah, Richmond; Aubyn, Edmond; Prah, James; Nwaefuna, Ekene Kwabena; Boampong, Johnson N

2016-01-01

The clinical outcome of falciparum malaria in endemic areas is influenced by erythrocyte polymorphisms including the ABO blood groups. Studies have reported association of ABO blood group to resistance, susceptibility, and severity of P. falciparum malaria infection. Individuals with blood group "A" have been found to be highly susceptible to falciparum malaria whereas blood group "O" is said to confer protection against complicated cases. We analyzed samples from 293 young children less than six years old with malaria in the Korle-Bu Teaching Hospital in Accra, Ghana. It was observed that group O was present in about 16.1% of complicated cases weighed against 40.9% of uncomplicated controls. Individuals with complicated malaria were about twice likely to be of blood groups A and B compared to group O (A versus O, OR = 1.90, 95% CI = 1.59-2.26, P < 0.0001; B versus O, OR = 1.82. 95% CI = 1.57-2.23, P < 0.0001). Blood group O participants with complicated diseases had low parasitaemia compared to the other blood groups (P < 0.0001). This may give blood group O individuals a survival advantage over the other groups in complicated malaria as suggested. Participants with complicated falciparum malaria were generally anaemic and younger than those with uncomplicated disease.

Trafficking of the signature protein of intra-erythrocytic Plasmodium berghei-induced structures, IBIS1, to P. falciparum Maurer's clefts.

PubMed

Petersen, Wiebke; Matuschewski, Kai; Ingmundson, Alyssa

2015-01-01

Remodeling of the host red blood cell by Plasmodium falciparum is well established and crucial for infection and parasite virulence. Host cell modifications are not exclusive to human Plasmodium parasites and also occur in hepatocytes and erythrocytes infected with murine Plasmodium parasites. The recently described intra-erythrocytic P. berghei-induced structures (IBIS) share similarities to P. falciparum Maurer's clefts. It is shown here that a potential candidate IBIS1 homologue in P. falciparum, PfHYP12 (PF3D7_1301400), is partially exported into the erythrocyte cytoplasm. To analyze a potential similarity between IBIS and Maurer's clefts we expressed the signature protein of IBIS in P. falciparum parasites. Visualization of the tagged protein revealed that PbIBIS1 can be exported by P. falciparum and localizes to Maurer's clefts in P. falciparum-infected erythrocytes, which indicates that IBIS and Maurer's clefts may be evolutionarily conserved parasite-induced structures in infected erythrocytes. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Native microflora in fresh-cut processing plants and their potentials of biofilm formation

USDA-ARS?s Scientific Manuscript database

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...

Differential T-cell responses to a chimeric Plasmodium falciparum antigen; UB05-09, correlates with acquired immunity to malaria.

PubMed

Dinga, J N; Njimoh, D L; Kiawa, B; Djikeng, A; Nyasa, R B; Nkuo-Akenji, T; Pellé, R; Titanji, V P K

2016-05-01

The development of a sterilizing and cost-effective vaccine against malaria remains a major problem despite recent advances. In this study, it is demonstrated that two antigens of P. falciparum UB05, UB09 and their chimera UB05-09 can serve as protective immunity markers by eliciting higher T-cell responses in malaria semi-immune subjects (SIS) than in frequently sick subjects (FSS) and could be used to distinguish these two groups. UB05, UB09 and UB05-09 were cloned, expressed in E. coli, purified and used to stimulate PBMCs isolated from 63 subjects in a malaria endemic area, for IFN-γ production, which was measured by the ELISpot assay. The polymorphism of UB09 gene in the malaria infected population was also studied by PCR/sequencing of the gene in P. falciparum field isolates. All three antigens were preferentially recognized by PBMCs from SIS. IFN-γ production induced by these antigens correlated with the absence of fever and parasitaemia. UB09 was shown to be relatively well-conserved in nature. It is concluded that UB05, UB09 and the chimera UB05-09 posses T-cell epitopes that are associated with protection against malaria and could thus be used to distinguish SIS from FSS eventhough acute infection with malaria has been shown to reduce cytokine production in some studies. Further investigations of these antigens as potential diagnostic and/or vaccine candidates for malaria are indicated. © 2016 John Wiley & Sons Ltd.

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

Spatial and space-time distribution of Plasmodium vivax and Plasmodium falciparum malaria in China, 2005-2014.

PubMed

Hundessa, Samuel H; Williams, Gail; Li, Shanshan; Guo, Jinpeng; Chen, Linping; Zhang, Wenyi; Guo, Yuming

2016-12-19

Despite the declining burden of malaria in China, the disease remains a significant public health problem with periodic outbreaks and spatial variation across the country. A better understanding of the spatial and temporal characteristics of malaria is essential for consolidating the disease control and elimination programme. This study aims to understand the spatial and spatiotemporal distribution of Plasmodium vivax and Plasmodium falciparum malaria in China during 2005-2009. Global Moran's I statistics was used to detect a spatial distribution of local P. falciparum and P. vivax malaria at the county level. Spatial and space-time scan statistics were applied to detect spatial and spatiotemporal clusters, respectively. Both P. vivax and P. falciparum malaria showed spatial autocorrelation. The most likely spatial cluster of P. vivax was detected in northern Anhui province between 2005 and 2009, and western Yunnan province between 2010 and 2014. For P. falciparum, the clusters included several counties of western Yunnan province from 2005 to 2011, Guangxi from 2012 to 2013, and Anhui in 2014. The most likely space-time clusters of P. vivax malaria and P. falciparum malaria were detected in northern Anhui province and western Yunnan province, respectively, during 2005-2009. The spatial and space-time cluster analysis identified high-risk areas and periods for both P. vivax and P. falciparum malaria. Both malaria types showed significant spatial and spatiotemporal variations. Contrary to P. vivax, the high-risk areas for P. falciparum malaria shifted from the west to the east of China. Further studies are required to examine the spatial changes in risk of malaria transmission and identify the underlying causes of elevated risk in the high-risk areas.

Reconstructing the Qo Site of Plasmodium falciparum bc 1 Complex in the Yeast Enzyme

PubMed Central

Vallières, Cindy; Fisher, Nicholas; Meunier, Brigitte

2013-01-01

The bc 1 complex of the mitochondrial respiratory chain is essential for Plasmodium falciparum proliferation, the causative agent of human malaria. Therefore, this enzyme is an attractive target for antimalarials. However, biochemical investigations of the parasite enzyme needed for the study of new drugs are challenging. In order to facilitate the study of new compounds targeting the enzyme, we are modifying the inhibitor binding sites of the yeast Saccharomyces cerevisiae to generate a complex that mimics the P. falciparum enzyme. In this study we focused on its Qo pocket, the site of atovaquone binding which is a leading antimalarial drug used in treatment and causal prophylaxis. We constructed and studied a series of mutants with modified Qo sites where yeast residues have been replaced by P. falciparum equivalents, or, for comparison, by human equivalents. Mitochondria were prepared from the yeast Plasmodium-like and human-like Qo mutants. We measured the bc 1 complex sensitivity to atovaquone, azoxystrobin, a Qo site targeting fungicide active against P. falciparum and RCQ06, a quinolone-derivative inhibitor of P. falciparum bc 1 complex.The data obtained highlighted variations in the Qo site that could explain the differences in inhibitor sensitivity between yeast, plasmodial and human enzymes. We showed that the yeast Plasmodium-like Qo mutants could be useful and easy-to-use tools for the study of that class of antimalarials. PMID:23951230

Simple Molecular Methods for Early Detection of Chloroquine Drug Resistance in Plasmodium vivax and Plasmodium falciparum

PubMed Central

Singh, Raksha; Urhehar, Anant Dattatraya

2016-01-01

Introduction Malaria is a human disease of which causes high morbidity and mortality. In Plasmodium falciparum malaria, the resistance to antimalarial drugs, especially chloroquine (CQ) is one of the paramount factors contributing to the global increase in morbidity and mortality, due to malaria. Hence, there is a need for detection of chloroquine drug resistance genes i.e., pfcrt-o (Plasmodium falciparum chloroquine resistance transporter-o) and pfmdr-1 (Plasmodium falciparum multidrug resistance-1) of P. falciparum and pvcrt-o (Plasmodium vivax chloroquine resistance transporter-o) and pvmdr-1 (Plasmodium vivax multidrug resistance-1) of P. vivax by using molecular methods to prevent mortality in malarial cases. Aim To standardize chloroquine drug sensitivity testing by molecular method so as to provide reports of chloroquine within 6-8 hours to physicians for better treatment. Materials and Methods This study was conducted over a period of one year from January to December 2014. A Total of 300 blood samples were collected from malaria suspected patient attending MGM Hospital, Kamothe, Navi Mumbai, India. Out of 300 blood samples, 44 were malaria positive as assessed by Thick and Thin blood smear stained, by Leishman’s method and examination with light microscope. Chloroquine drug sensitivity testing was performed using WHO III plate method (micro test). Nested PCR was done for detection of pfcrt-o and pfmdr-1 for P. falciparum and pvcrt-o, pvmdr-1 genes for P. vivax. Results Total 44 samples were included in this study, out of which 22 samples confirmed for Plasmodium falciparum and 22 samples confirmed for Plasmodium vivax. Out of 22 P. falciparum 15 (68.18%) samples were chloroquine resistant. P. vivax showed chloroquine resistance to 5 samples (22.73%) by method similar to WHO III plate method (micro test) and nested PCR. Conclusion Drug resistance testing by molecular methods is useful for early detection of antimalarial drug resistance. pfmdr-1 along with

Sequence homology and structural analysis of plasmepsin 4 isolated from Indian Plasmodium vivax isolates.

PubMed

Rawat, Manmeet; Vijay, Sonam; Gupta, Yash; Dixit, Rajnikant; Tiwari, P K; Sharma, Arun

2011-07-01

Plasmodium vivax malaria is a globally widespread disease responsible for 50% of human malaria cases in Central and South America, South East Asia and Indian subcontinent. The rising severity of the disease and emerging resistance of the parasite has emphasized the need for the search of novel therapeutic targets to combat P. vivax malaria. Plasmepsin 4 (PM4) a food vacuole aspartic protease is essential in parasite functions and viability such as initiating hemoglobin digestion and processing of proteins and is being looked upon as potential drug target. Although the plasmepsins of Plasmodium falciparum have been extensively studied, the plasmepsins of P. vivax are not well characterized. This is the first report detailing complete PM4 gene analysis from Indian P. vivax isolates. Blast results of sequences of P. vivax plasmepsin 4 (PvPM4) shows 100% homology among isolates of P. vivax collected from different geographical regions of India. All of the seven Indian isolates did not contain intron within the coding region. Interestingly, PvPM4 sequence analysis showed a very high degree of homology with all other sequences of Plasmodium species available in the genebank. Our results strongly suggest that PvPM4 are highly conserved except a small number of amino acid substitutions that did not modify key motifs at active site formation for the function or the structure of the enzymes. Furthermore, our study shows that PvPM4 occupies unique phylogenetic status within Plasmodium group and sufficiently differ from the most closely related human aspartic protease, cathepsin D. The analysis of 3D model of PM4 showed a typical aspartic protease structure with bi-lobed, compact and distinct peptide binding cleft in both P. vivax and P. falciparum. In order to validate appropriate use of PM4 as potential anti-malarial drug target, studies on genetic and structural variations among P. vivax plasmepsins (PvPMs) from different geographical regions are of utmost importance for

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. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.

Effects of larvicidal and larval nutritional stresses on Anopheles gambiae development, survival and competence for Plasmodium falciparum.

PubMed

Vantaux, Amélie; Ouattarra, Issiaka; Lefèvre, Thierry; Dabiré, Kounbobr Roch

2016-04-23

Many studies have shown that the environment in which larvae develop can influence adult characteristics with consequences for the transmission of pathogens. We investigated how two environmental stresses (larviciding and nutritional stress) interact to affect Anopheles gambiae (previously An. gambiae S molecular form) life history traits and its susceptibility for field isolates of its natural malaria agent Plasmodium falciparum. Larvae were reared in the presence or not of a sub-lethal concentration of larvicide and under a high and low food regimen. Development time, individual size, adult survival and competence for P. falciparum were assessed. Individuals under low food regimen took more time to develop, had a lower development success and were smaller while there was no main effect of larvicide exposure on these traits. However, larvicide exposure impacted individual size in interaction with nutritional stress. Female survival was affected by the interaction between gametocytemia, parasite exposure and larval diet, as well as the interaction between gametocytemia, parasite exposure and larvicidal stress, and the interaction between gametocytemia, larvicidal exposure and larval diet. Among the 951 females dissected 7 days post-infection, 559 (58.78%) harboured parasites. Parasite prevalence was significantly affected by the interaction between larvicidal stress and larval diet. Indeed, females under low food regimen had a higher prevalence than females under high food regimen and this difference was greater under larvicidal stress. The two stresses did not impact parasite intensity. We found that larval nutritional and larvicidal stresses affect mosquito life history traits in complex ways, which could greatly affect P. falciparum transmission. Further studies combining field-based trials on larvicide use and mosquito experimental infections would give a more accurate understanding of the effects of this vector control tool on malaria transmission.

Genome-wide analysis of selection on the malaria parasite Plasmodium falciparum in West African populations of differing infection endemicity.

PubMed

Mobegi, Victor A; Duffy, Craig W; Amambua-Ngwa, Alfred; Loua, Kovana M; Laman, Eugene; Nwakanma, Davis C; MacInnis, Bronwyn; Aspeling-Jones, Harvey; Murray, Lee; Clark, Taane G; Kwiatkowski, Dominic P; Conway, David J

2014-06-01

Locally varying selection on pathogens may be due to differences in drug pressure, host immunity, transmission opportunities between hosts, or the intensity of between-genotype competition within hosts. Highly recombining populations of the human malaria parasite Plasmodium falciparum throughout West Africa are closely related, as gene flow is relatively unrestricted in this endemic region, but markedly varying ecology and transmission intensity should cause distinct local selective pressures. Genome-wide analysis of sequence variation was undertaken on a sample of 100 P. falciparum clinical isolates from a highly endemic region of the Republic of Guinea where transmission occurs for most of each year and compared with data from 52 clinical isolates from a previously sampled population from The Gambia, where there is relatively limited seasonal malaria transmission. Paired-end short-read sequences were mapped against the 3D7 P. falciparum reference genome sequence, and data on 136,144 single nucleotide polymorphisms (SNPs) were obtained. Within-population analyses identifying loci showing evidence of recent positive directional selection and balancing selection confirm that antimalarial drugs and host immunity have been major selective agents. Many of the signatures of recent directional selection reflected by standardized integrated haplotype scores were population specific, including differences at drug resistance loci due to historically different antimalarial use between the countries. In contrast, both populations showed a similar set of loci likely to be under balancing selection as indicated by very high Tajima's D values, including a significant overrepresentation of genes expressed at the merozoite stage that invades erythrocytes and several previously validated targets of acquired immunity. Between-population FST analysis identified exceptional differentiation of allele frequencies at a small number of loci, most markedly for five SNPs covering a 15-kb

Associations Between Helminth Infections, Plasmodium falciparum Parasite Carriage and Antibody Responses to Sexual and Asexual Stage Malarial Antigens.

PubMed

Ateba-Ngoa, Ulysse; Jones, Sophie; Zinsou, Jeannot Fréjus; Honkpehedji, Josiane; Adegnika, Ayola Akim; Agobe, Jean-Claude Dejon; Massinga-Loembe, Marguerite; Mordmüller, Benjamin; Bousema, Teun; Yazdanbakhsh, Maria

2016-08-03

Infections with helminths and Plasmodium spp. overlap in their geographical distribution. It has been postulated that helminth infections may influence malarial transmission by altering Plasmodium falciparum gametocytogenesis. This cross-sectional study assessed the effect of helminth infections on P. falciparum gametocyte carriage and on humoral immune responses to sexual stage antigens in Gabon. Schistosoma haematobium and filarial infections as well as P. falciparum asexual forms and gametocyte carriage were determined. The antibody responses measured were to sexual (Pfs230, Pfs48/45) and asexual P. falciparum antigens (AMA1, MSP1, and GLURP). A total of 287 subjects were included. The prevalence of microscopically detectable P. falciparum asexual parasites was higher in S. haematobium-infected subjects in comparison to their uninfected counterparts (47% versus 26%, P = 0.003), but this was not different when filarial infections were considered. Plasmodium falciparum gametocyte carriage was similar between Schistosoma- or filaria-infected and uninfected subjects. We observed a significant decrease of Pfs48/45 immunoglobulin G titer in S. haematobium-infected subjects (P = 0.037), whereas no difference was seen for Pfs230 antibody titer, nor for antibodies to AMA1, MSP1, or GLURP. Our findings suggest an effect of S. haematobium on antibody responses to some P. falciparum gametocyte antigens that may have consequences for transmission-blocking immunity. © The American Society of Tropical Medicine and Hygiene.

Case Report: A Case of Plasmodium falciparum hrp2 and hrp3 Gene Mutation in Bangladesh.

PubMed

Nima, Maisha Khair; Hougard, Thomas; Hossain, Mohammad Enayet; Kibria, Mohammad Golam; Mohon, Abu Naser; Johora, Fatema Tuj; Rahman, Rajibur; Haque, Rashidul; Alam, Mohammad Shafiul

2017-10-01

Several species of Plasmodium are responsible for causing malaria in humans. Proper diagnoses are crucial to case management, because severity and treatment varies between species. Diagnoses can be made using rapid diagnostic tests (RDTs), which detect Plasmodium proteins. Plasmodium falciparum causes the most virulent cases of malaria, and P. falciparum histidine-rich protein 2 (PfHRP2) is a common target of falciparum malaria RDTs. Here we report a case in which a falciparum malaria patient in Bangladesh tested negative on PfHRP2-based RDTs. The negative results can be attributed to a deletion of part of the pfhrp2 gene and frameshift mutations in both pfhrp2 and pfhrp3 gene. This finding may have implications for malaria diagnostics and case management in Bangladesh and other regions of South Asia.

Plasmodium falciparum Genetic Diversity in Bangladesh Does Not Suggest a Hypoendemic Population Structure

PubMed Central

Alam, Mohammad Shafiul; Elahi, Rubayet; Mohon, Abu Naser; Al-Amin, Hasan Mohammad; Kibria, Mohammad Golam; Khan, Wasif A.; Khanum, Hamida; Haque, Rashidul

2016-01-01

Despite the recommendation for the use of merozoite surface protein 1 (msp1), merozoite surface protein 2 (msp2), and glutamate-rich protein (glurp) genes as markers in drug efficacy studies by World Health Organization and their limited use in Bangladesh, the circulating Plasmodium falciparum population genetic structure has not yet been assessed in Bangladesh. This study presents a comprehensive report on the circulating P. falciparum population structure based on msp1, msp2, and glurp polymorphic gene markers in Bangladesh. Among the 130 pretreatment (day 0) P. falciparum samples from seven malaria-endemic districts, 14 distinct genotypes were observed for msp1, 20 for msp2, and 13 for glurp. Polyclonal infection was reported in 94.6% (N = 123) of the samples. Multiplicity of infection (MOI) for msp1 was the highest (1.5) among the MOIs of the markers. The heterozygosity for msp1, msp2, and glurp was 0.89, 0.93, and 0.83, respectively. Data according to different malaria-endemic areas are also presented and discussed. Bangladesh is considered as a malaria-hypoendemic country. However, the prevalence of polyclonal infection and the genetic diversity of P. falciparum do not represent hypoendemicity. PMID:27139455

Resistance screening and trend analysis of imported falciparum malaria in NSW, Australia (2010 to 2016).

PubMed

Prosser, Christiane; Meyer, Wieland; Ellis, John; Lee, Rogan

2018-01-01

The World Health Organization currently recommends artemisinin (along with a partner drug) as the global frontline treatment for Plasmodium falciparum malaria. Artemisinin resistant P. falciparum are now found throughout the greater Mekong subregion of South East Asia. Several polymorphisms in the parasite's kelch gene have been demonstrated to confer artemisinin resistance. While genotypes within the greater Mekong subregion are thoroughly examined in the literature, P. falciparum populations within several areas that do not (yet) have endemic resistance are underrepresented. This investigation characterised the Pfkelch13 propeller domains from 153 blood samples of 140 imported cases of P. falciparum malaria in New South Wales from 2010 to 2016. A low level of propeller domain diversity was observed, including the C580Y coding mutation most strongly associated with artemisinin resistance in South East Asia. The resistance genotype was found in a sample originating in Papua New Guinea, where this mutation, or artemisinin treatment failure, have not been previously reported. Sequencing a panel of geographically informative polymorphisms within the organellar genomes identified the C580Y parasite as having Oceanic origins. Patient data analysis revealed that New South Wales, Australia, P. falciparum malaria cases often originated from regions with limited drug resistance screening. The C580Y finding from outside of the greater Mekong subregion supports the consensus to upscale molecular surveillance of artemisinin resistance outside of South East Asia. The genetic screening results identify a risk of importing resistant falciparum malaria to Australia, supporting an ongoing surveillance protocol to pre-empt treatment failure and contribute to global data gathering.

Genome-Level Determination of Plasmodium falciparum Blood-Stage Targets of Malarial Clinical Immunity in the Peruvian Amazon

PubMed Central

Torres, Katherine J.; Castrillon, Carlos E.; Moss, Eli L.; Saito, Mayuko; Tenorio, Roy; Molina, Douglas M.; Davies, Huw; Neafsey, Daniel E.; Felgner, Philip; Vinetz, Joseph M.; Gamboa, Dionicia

2015-01-01

Background. Persons with blood-stage Plasmodium falciparum parasitemia in the absence of symptoms are considered to be clinically immune. We hypothesized that asymptomatic subjects with P. falciparum parasitemia would differentially recognize a subset of P. falciparum proteins on a genomic scale. Methods and Findings. Compared with symptomatic subjects, sera from clinically immune, asymptomatically infected individuals differentially recognized 51 P. falciparum proteins, including the established vaccine candidate PfMSP1. Novel, hitherto unstudied hypothetical proteins and other proteins not previously recognized as potential vaccine candidates were also differentially recognized. Genes encoding the proteins differentially recognized by the Peruvian clinically immune individuals exhibited a significant enrichment of nonsynonymous nucleotide variation, an observation consistent with these genes undergoing immune selection. Conclusions. A limited set of P. falciparum protein antigens was associated with the development of naturally acquired clinical immunity in the low-transmission setting of the Peruvian Amazon. These results imply that, even in a low-transmission setting, an asexual blood-stage vaccine designed to reduce clinical malaria symptoms will likely need to contain large numbers of often-polymorphic proteins, a finding at odds with many current efforts in the design of vaccines against asexual blood-stage P. falciparum. PMID:25381370

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.

Antimicrobial activity of fresh garlic juice: An in vitro study

PubMed Central

Yadav, Seema; Trivedi, Niyati A.; Bhatt, Jagat D.

2015-01-01

Introduction: Antimicrobial resistance has been a global concern. Currently, interest has been focused on exploring antimicrobial properties of plants and herbs. One such botanical is Allium sativum (garlic). Aim: To evaluate the antimicrobial activity of fresh juice of garlic. Materials and Methods: Varying concentrations of fresh garlic juice (FGJ) were tested for their antimicrobial activity against common pathogenic organisms isolated at SSG Hospital, Vadodara, using well diffusion method. Moreover, minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) of FGJ were tested using broth dilution method. Sensitivity pattern of the conventional antimicrobials against common pathogenic bacteria was tested using disc diffusion method. Results: FGJ produced dose-dependent increase in the zone of inhibition at a concentration of 10% and higher. MIC of FGJ against the pathogens ranged from 4% to 16% v/v whereas MLC value ranged from 4% to 32% v/v with Escherichia coli and Staphylococcus aureus spp. showed highest sensitivity. Conclusion: FGJ has definite antimicrobial activity against common pathogenic organisms isolated at SSG Hospital, Vadodara. Further studies are needed to find out the efficacy, safety, and kinetic data of its active ingredients. PMID:27011724

Rapid diagnostic tests for diagnosing uncomplicated non-falciparum or Plasmodium vivax malaria in endemic countries

PubMed Central

Abba, Katharine; Kirkham, Amanda J; Olliaro, Piero L; Deeks, Jonathan J; Donegan, Sarah; Garner, Paul; Takwoingi, Yemisi

2014-01-01

Background In settings where both Plasmodium vivax and Plasmodium falciparum infection cause malaria, rapid diagnostic tests (RDTs) need to distinguish which species is causing the patients' symptoms, as different treatments are required. Older RDTs incorporated two test lines to distinguish malaria due to P. falciparum, from malaria due to any other Plasmodium species (non-falciparum). These RDTs can be classified according to which antibodies they use: Type 2 RDTs use HRP-2 (for P. falciparum) and aldolase (all species); Type 3 RDTs use HRP-2 (for P. falciparum) and pLDH (all species); Type 4 use pLDH (fromP. falciparum) and pLDH (all species). More recently, RDTs have been developed to distinguish P. vivax parasitaemia by utilizing a pLDH antibody specific to P. vivax. Objectives To assess the diagnostic accuracy of RDTs for detecting non-falciparum or P. vivax parasitaemia in people living in malaria-endemic areas who present to ambulatory healthcare facilities with symptoms suggestive of malaria, and to identify which types and brands of commercial test best detect non-falciparum and P. vivax malaria. Search methods We undertook a comprehensive search of the following databases up to 31 December 2013: Cochrane Infectious Diseases Group Specialized Register; MEDLINE; EMBASE; MEDION; Science Citation Index; Web of Knowledge; African Index Medicus; LILACS; and IndMED. Selection criteria Studies comparing RDTs with a reference standard (microscopy or polymerase chain reaction) in blood samples from a random or consecutive series of patients attending ambulatory health facilities with symptoms suggestive of malaria in non-falciparum endemic areas. Data collection and analysis For each study, two review authors independently extracted a standard set of data using a tailored data extraction form. We grouped comparisons by type of RDT (defined by the combinations of antibodies used), and combined in meta-analysis where appropriate. Average sensitivities and

A global transcriptional analysis of Plasmodium falciparum malaria reveals a novel family of telomere-associated lncRNAs

PubMed Central

2011-01-01

Background Mounting evidence suggests a major role for epigenetic feedback in Plasmodium falciparum transcriptional regulation. Long non-coding RNAs (lncRNAs) have recently emerged as a new paradigm in epigenetic remodeling. We therefore set out to investigate putative roles for lncRNAs in P. falciparum transcriptional regulation. Results We used a high-resolution DNA tiling microarray to survey transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. We identified 872 protein-coding genes and 60 putative P. falciparum lncRNAs under developmental regulation during the parasite's pathogenic human blood stage. Further characterization of lncRNA candidates led to the discovery of an intriguing family of lncRNA telomere-associated repetitive element transcripts, termed lncRNA-TARE. We have quantified lncRNA-TARE expression at 15 distinct chromosome ends and mapped putative transcriptional start and termination sites of lncRNA-TARE loci. Remarkably, we observed coordinated and stage-specific expression of lncRNA-TARE on all chromosome ends tested, and two dominant transcripts of approximately 1.5 kb and 3.1 kb transcribed towards the telomere. Conclusions We have characterized a family of 22 telomere-associated lncRNAs in P. falciparum. Homologous lncRNA-TARE loci are coordinately expressed after parasite DNA replication, and are poised to play an important role in P. falciparum telomere maintenance, virulence gene regulation, and potentially other processes of parasite chromosome end biology. Further study of lncRNA-TARE and other promising lncRNA candidates may provide mechanistic insight into P. falciparum transcriptional regulation. PMID:21689454

The Hsp40 proteins of Plasmodium falciparum and other apicomplexa: regulating chaperone power in the parasite and the host.

PubMed

Botha, M; Pesce, E-R; Blatch, G L

2007-01-01

Extensive structural and functional remodelling of Plasmodium falciparum (malaria)-infected erythrocytes follows the export of a range of proteins of parasite origin (exportome) across the parasitophorous vacuole into the host erythrocyte. The genome of P. falciparum encodes a diverse chaperone complement including at least 43 members of the heat shock protein 40kDa (Hsp40) family, and six members of the heat shock protein 70kDa (Hsp70) family. Nearly half of the Hsp40 proteins of P. falciparum are predicted to contain a PEXEL/HT (Plasmodium export element/host targeting signal) sequence motif, and hence are likely to be part of the exportome. In this review we critically evaluate the classification, sequence similarity and clustering, and possible interactors of the P. falciparum Hsp40 chaperone machinery. In addition to the types I, II and III Hsp40 proteins all exhibiting the signature J-domain, the P. falciparum genome also encodes a number of specialized Hsp40 proteins with a J-like domain, which we have categorized as type IV Hsp40 proteins. Analysis of the potential P. falciparum Hsp40 protein interaction network revealed connections predominantly with cytoskeletal and membrane proteins, transcriptional machinery, DNA repair and replication machinery, translational machinery, the proteasome and proteolytic enzymes, and enzymes involved in cellular physiology. Comparison of the Hsp40 proteins of P. falciparum to those of other apicomplexa reveals that most of the proteins (especially the PEXEL/HT-containing proteins) are unique to P. falciparum. Furthermore, very few of the P. falciparum Hsp40 proteins have human homologs, except for those proteins implicated in fundamental biological processes. Our analysis suggests that P. falciparum has evolved an expanded and specialized Hsp40 protein machinery to enable it successfully to invade and remodel the human erythrocyte, and we propose a model in which these proteins are involved in chaperone

Evaluation of Ebola virus inactivation procedures for Plasmodium falciparum malaria diagnostics.

PubMed

Lau, Rachel; Wang, Amanda; Chong-Kit, Ann; Ralevski, Filip; Boggild, Andrea K

2015-04-01

Plasmodium falciparum malaria is highly endemic in the three most affected countries in the current epidemic of Ebola virus disease (EVD) in West Africa. As EVD and malaria are clinically indistinguishable, both remain part of the differential diagnosis of ill travelers from returning from areas of EVD transmission. We compared the performances of a rapid diagnostic test (BinaxNOW) and real-time PCR with P. falciparum-positive specimens before and after heat and Triton X-100 inactivation, and we documented no loss of sensitivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A new world malaria map: Plasmodium falciparum endemicity in 2010.

PubMed

Gething, Peter W; Patil, Anand P; Smith, David L; Guerra, Carlos A; Elyazar, Iqbal R F; Johnston, Geoffrey L; Tatem, Andrew J; Hay, Simon I

2011-12-20

Transmission intensity affects almost all aspects of malaria epidemiology and the impact of malaria on human populations. Maps of transmission intensity are necessary to identify populations at different levels of risk and to evaluate objectively options for disease control. To remain relevant operationally, such maps must be updated frequently. Following the first global effort to map Plasmodium falciparum malaria endemicity in 2007, this paper describes the generation of a new world map for the year 2010. This analysis is extended to provide the first global estimates of two other metrics of transmission intensity for P. falciparum that underpin contemporary questions in malaria control: the entomological inoculation rate (PfEIR) and the basic reproductive number (PfR). Annual parasite incidence data for 13,449 administrative units in 43 endemic countries were sourced to define the spatial limits of P. falciparum transmission in 2010 and 22,212 P. falciparum parasite rate (PfPR) surveys were used in a model-based geostatistical (MBG) prediction to create a continuous contemporary surface of malaria endemicity within these limits. A suite of transmission models were developed that link PfPR to PfEIR and PfR and these were fitted to field data. These models were combined with the PfPR map to create new global predictions of PfEIR and PfR. All output maps included measured uncertainty. An estimated 1.13 and 1.44 billion people worldwide were at risk of unstable and stable P. falciparum malaria, respectively. The majority of the endemic world was predicted with a median PfEIR of less than one and a median PfRc of less than two. Values of either metric exceeding 10 were almost exclusive to Africa. The uncertainty described in both PfEIR and PfR was substantial in regions of intense transmission. The year 2010 has a particular significance as an evaluation milestone for malaria global health policy. The maps presented here contribute to a rational basis for control and

Plasmodium falciparum-induced severe malaria with acute kidney injury and jaundice: a case report

NASA Astrophysics Data System (ADS)

Baswin, A.; Siregar, M. L.; Jamil, K. F.

2018-03-01

P. falciparum-induced severe malaria with life-threatening complications like acute kidney injury (AKI), jaundice, cerebral malaria, severe anemia, acidosis, and acute respiratory distress syndrome (ARDS). A 31-year-old soldier man who works in Aceh Singkil, Indonesia which is an endemic malaria area presented with a paroxysm of fever, shaking chills and sweats over four days, headache, arthralgia, abdominal pain, pale, jaundice, and oliguria. Urinalysis showed hemoglobinuria. Blood examination showed hemolytic anemia, thrombocytopenia, and hyperbilirubinemia. Falciparum malaria was then confirmed by peripheral blood smear, antimalarial medications were initiated, and hemodialysis was performed for eight times. The patient’s condition and laboratory results were quickly normalized. We report a case of P. falciparum-induced severe malaria with AKI and jaundice. The present case suggests that P. falciparum may induce severe malaria with life-threatening complications, early diagnosis and treatment is important to improve the quality of life of patients. Physicians must be alert for correct diagnosis and proper management of imported tropical malaria when patients have travel history in endemic areas.

Limonene Arrests Parasite Development and Inhibits Isoprenylation of Proteins in Plasmodium falciparum

PubMed Central

Moura, Ivan Cruz; Wunderlich, Gerhard; Uhrig, Maria L.; Couto, Alicia S.; Peres, Valnice J.; Katzin, Alejandro M.; Kimura, Emília A.

2001-01-01

Isoprenylation is an essential protein modification in eukaryotic cells. Herein, we report that in Plasmodium falciparum, a number of proteins were labeled upon incubation of intraerythrocytic forms with either [3H]farnesyl pyrophosphate or [3H]geranylgeranyl pyrophosphate. By thin-layer chromatography, we showed that attached isoprenoids are partially modified to dolichol and other, uncharacterized, residues, confirming active isoprenoid metabolism in this parasite. Incubation of blood-stage P. falciparum treated with the isoprenylation inhibitor limonene significantly decreased the parasites' progression from the ring stage to the trophozoite stage and at 1.22 mM, 50% of the parasites died after the first cycle. Using Ras- and Rap-specific monoclonal antibodies, putative Rap and Ras proteins of P. falciparum were immunoprecipitated. Upon treatment with 0.5 mM limonene, isoprenylation of these proteins was significantly decreased, possibly explaining the observed arrest of parasite development. PMID:11502528

Purification and partial characterization of PfHRP-II protein of Plasmodium falciparum.

PubMed

Ghimire, Prakash; Samantaray, J C; Mirdha, B R; Patra, A K; Panda, A K

2003-12-01

The human malarial parasite Plasmodium falciparum secretes various intra-and extra-cellular proteins during its asexual life cycle in human RBC. Histidine rich protein-II (HRP-II) is one of the most prominent proteins, found to be secreted by P. falciparum throughout the asexual cycle with the peak during mature schizont stage of the parasite development in human IRBC. The high histidine content (35% of the total amino acids in protein) of this protein suggested the potential to bind divalent metal ions. We have demonstrated by metal chelate chromatography, an extraordinary capacity of HRP-II to bind nickel ions (Ni++) and employed this characteristic to purify the extra-cellular HRP-II protein secreted by P. falciparum from culture supernatant. The identity of the purified protein was verified by the relative molecular weight on SDS-PAGE, by reacting with polyclonal antibodies directed against it using Western blot technique.

Estimating the parasitaemia of Plasmodium falciparum: experience from a national EQA scheme

PubMed Central

2013-01-01

Background To examine performance of the identification and estimation of percentage parasitaemia of Plasmodium falciparum in stained blood films distributed in the UK National External Quality Assessment Scheme (UKNEQAS) Blood Parasitology Scheme. Methods Analysis of performance for the diagnosis and estimation of the percentage parasitaemia of P. falciparum in Giemsa-stained thin blood films was made over a 15-year period to look for trends in performance. Results An average of 25% of participants failed to estimate the percentage parasitaemia, 17% overestimated and 8% underestimated, whilst 5% misidentified the malaria species present. Conclusions Although the results achieved by participants for other blood parasites have shown an overall improvement, the level of performance for estimation of the parasitaemia of P. falciparum remains unchanged over 15 years. Possible reasons include incorrect calculation, not examining the correct part of the film and not examining an adequate number of microscope fields. PMID:24261625

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). Copyright © 2015, Ochola-Oyier et al.

The Exported Chaperone PfHsp70x Is Dispensable for the Plasmodium falciparum Intraerythrocytic Life Cycle.

PubMed

Cobb, David W; Florentin, Anat; Fierro, Manuel A; Krakowiak, Michelle; Moore, Julie M; Muralidharan, Vasant

2017-01-01

Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum during its asexual life cycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by P. falciparum in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70, P. falciparum Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most Plasmodium species and is found only in P. falciparum and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in P. falciparum to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of P. falciparum . IMPORTANCE Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite's ability to survive within the erythrocyte is

Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum*

PubMed Central

Yam, Xue Yan; Birago, Cecilia; Fratini, Federica; Di Girolamo, Francesco; Raggi, Carla; Sargiacomo, Massimo; Bachi, Angela; Berry, Laurence; Fall, Gamou; Currà, Chiara; Pizzi, Elisabetta; Breton, Catherine Braun; Ponzi, Marta

2013-01-01

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in

Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia

PubMed Central

Mkulama, Mtawa AP; Chishimba, Sandra; Sikalima, Jay; Rouse, Petrica; Thuma, Philip E; Mharakurwa, Sungano

2008-01-01

Background In Zambia the first-line treatment for uncomplicated malaria is artemisinin combination therapy (ACT), with artemether-lumefantrine currently being used. However, the antifolate regimen, sulphadoxine-pyrimethamine (SP), remains the treatment of choice in children weighing less than 5 kg and also in expectant mothers. SP is also the choice drug for intermittent preventive therapy in pregnancy and serves as stand-by treatment during ACT stock outs. The current study assessed the status of Plasmodium falciparum point mutations associated with antifolate drug resistance in the area around Macha. Methods A representative sample of 2,780 residents from the vicinity of Macha was screened for malaria by microscopy. At the same time, blood was collected onto filter paper and dried for subsequent P. falciparum DNA analysis. From 188 (6.8%) individuals that were thick film-positive, a simple random sub-set of 95 P. falciparum infections were genotyped for DHFR and DHPS antifolate resistance mutations, using nested PCR and allele-specific restriction enzyme digestion. Results Plasmodium falciparum field samples exhibited a high prevalence of antifolate resistance mutations, including the DHFR triple (Asn-108 + Arg-59 + Ile-51) mutant (41.3%) and DHPS double (Gly-437 + Glu-540) mutant (16%). The quintuple (DHFR triple + DHPS double) mutant was found in 4 (6.5%) of the samples. Levels of mutated parasites showed a dramatic escalation, relative to previous surveys since 1988. However, neither of the Val-16 and Thr-108 mutations, which jointly confer resistance to cycloguanil, was detectable among the human infections. The Leu-164 mutation, associated with high grade resistance to both pyrimethamine and cycloguanil, as a multiple mutant with Asn-108, Arg-59 and (or) Ile-51, was also absent. Conclusion This study points to escalating levels of P. falciparum antifolate resistance in the vicinity of Macha. Continued monitoring is recommended to ensure timely policy

Impairment of the Plasmodium falciparum erythrocytic cycle induced by angiotensin peptides.

PubMed

Saraiva, Victor Barbosa; de Souza Silva, Leandro; Ferreira-DaSilva, Claudio Teixeira; da Silva-Filho, João Luiz; Teixeira-Ferreira, André; Perales, Jonas; Souza, Mariana Conceição; Henriques, Maria das Graças; Caruso-Neves, Celso; de Sá Pinheiro, Ana Acacia

2011-02-18

Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1-7). Parasite infection decreased Ang-(1-7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1-7) decreased the level of infection in an A779 (specific antagonist of Ang-(1-7) receptor, MAS)-sensitive manner. 10(-7) M PD123319, an AT(2) receptor antagonist, partially reversed the effects of Ang-(1-7) and Ang II. However, 10(-6) M losartan, an antagonist of the AT(1) receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10(-8) M Ang II or 10(-8) M Ang-(1-7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10(-7) M A779. 10(-6) M dibutyryl-cAMP increased the level of infection and 10(-7) M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1-7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1-7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus.

Efficacy of monthly tafenoquine for prophylaxis of Plasmodium vivax and multidrug-resistant P. falciparum malaria.

PubMed

Walsh, Douglas S; Eamsila, Chirapa; Sasiprapha, Theerayuth; Sangkharomya, Suebpong; Khaewsathien, Pradith; Supakalin, Panpaka; Tang, Douglas B; Jarasrumgsichol, Phongsak; Cherdchu, Chainarong; Edstein, Michael D; Rieckmann, Karl H; Brewer, Thomas G

2004-10-15

We assessed monthly doses of tafenoquine for preventing Plasmodium vivax and multidrug-resistant P. falciparum malaria. In a randomized, double-blind, placebo-controlled study, 205 Thai soldiers received either a loading dose of tafenoquine 400 mg (base) daily for 3 days, followed by single monthly 400-mg doses (n = 104), or placebo (n = 101), for up to 5 consecutive months. In volunteers completing follow-up (96 tafenoquine and 91 placebo recipients), there were 22 P. vivax, 8 P. falciparum, and 1 mixed infection. All infections except 1 P. vivax occurred in placebo recipients, giving tafenoquine a protective efficacy of 97% for all malaria (95% confidence interval [CI], 82%-99%), 96% for P. vivax malaria (95% CI, 76%-99%), and 100% for P. falciparum malaria (95% CI, 60%-100%). Monthly tafenoquine was safe, well tolerated, and highly effective in preventing P. vivax and multidrug-resistant P. falciparum malaria in Thai soldiers during 6 months of prophylaxis. Copyright 2004 Infectious Diseases Society of America

Clinical trials of artesunate plus sulfadoxine-pyrimethamine for Plasmodium falciparum malaria in Afghanistan: maintained efficacy a decade after introduction.

PubMed

Awab, Ghulam Rahim; Imwong, Mallika; Pukrittayakamee, Sasithon; Alim, Fazel; Hanpithakpong, Warunee; Tarning, Joel; Dondorp, Arjen M; Day, Nicholas P J; White, Nicholas J; Woodrow, Charles J

2016-02-25

Combination therapy with artesunate plus sulfadoxine-pyrimethamine (SP) was adopted as recommended treatment for Plasmodium falciparum infection in Afghanistan in 2003. A series of prospective clinical studies examining the efficacy of artesunate plus sulfadoxine-pyrimethamine (AS + SP) against P. falciparum were undertaken in sentinel sites in Afghanistan from 2007 to 2014, accompanied by relevant molecular studies. The first study was a randomized trial of AS + SP versus dihydroartemisinin-piperaquine, while two subsequent studies were standard therapeutic efficacy studies of AS + SP. Three hundred and three patients were enrolled across four provinces in the north and east of the country. Curative efficacy was high in all the trials, with an adequate clinical and parasitological response (ACPR) of more than 95 % in all groups and trial stages. Genotyping for drug-resistance alleles at dhfr indicated fixation of the S108 N mutation and a prevalence of the C59R mutation of approximately 95 % across all sites. Other mutations in dhfr and dhps remained rare or absent entirely, although five isolates from the first trial carried the dhps triple mutant SGEGA haplotype. In the last study undertaken in 2012-2014 the K13 artemisinin resistance marker was examined; only two of 60 successfully sequenced samples carried a K13-propeller mutation. These data confirm maintained efficacy 10 years after introduction of artesunate plus SP as combination treatment of P. falciparum in Afghanistan. The molecular data indicate that despite a substantial fall in incidence, resistance has not developed to artemisinins, or intensified to the ACT partner drug components. Trial Registration http://www.clinicaltrials.gov/ct NCT00682578, NCT01115439 and NCT01707199.

Imported Malaria in Turkey: The Importance of Diagnosis and Treatment of Plasmodium falciparum/Plasmodium vivax Mixed Infection.

PubMed

Tünger, Özlem; Çakmak, Akide; Özbilgin, Ahmet; Tunalı, Varol; Çetin, Çiğdem Banu

2018-05-21

The most common types of malaria in the world are Plasmodium vivax and P. falciparum. In countries where both species are endemic, P. vivax and P. falciparum coinfection also occurs. Thus, the possibility of mixed malaria in Turkey should always be considered in cases with a traveling history to these countries. Here, we report a case of P. vivax/P. falciparum mixed infection that was diagnosed as P. falciparum malaria in Ethiopia. However, the administered treatment was inadequate, and infection recurred because of the miss in the diagnosis of P. vivax malaria, for which an effective drug for hypnozoites was not administered. This case report emphasizes the importance of diagnosis, correct and adequate treatment of infections, and a close follow-up of diseases.

Development of loop-mediated isothermal amplification with Plasmodium falciparum unique genes for molecular diagnosis of human malaria.

PubMed

Zhang, Yijing; Yao, Yi; Du, Weixing; Wu, Kai; Xu, Wenyue; Lin, Min; Tan, Huabing; Li, Jian

2017-07-01

In order to achieve better outcomes for treatment and in the prophylaxis of malaria, it is imperative to develop a sensitive, specific, and accurate assay for early diagnosis of Plasmodium falciparum infection, which is the major cause of malaria. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay with P. falciparum unique genes for sensitive, specific, and accurate detection of P. falciparum infection. The unique genes of P. falciparum were randomly selected from PlasmoDB. The LAMP primers of the unique genes were designed using PrimerExplorer V4. LAMP assays with primers from unique genes of P. falciparum and conserved 18S rRNA gene were developed and their sensitivity was assessed. The specificity of the most sensitive LAMP assay was further examined using genomic DNA from Plasmodium vivax, Plasmodium yoelii and Toxoplasma gondii. Finally, the unique gene-based LAMP assay was validated using clinical samples of P. falciparum infection cases. A total of 31 sets of top-scored LAMP primers from nine unique genes were selected from the pools of designed primers. The LAMP assay with PF3D7_1253300-5 was the most sensitive with the detection limit 5 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. vivax, P. yoelii, and T. gondii. The LAMP assay with PF3D7_0112300 (18S rRNA) was less sensitive with the detection limit 50 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. yoelii and T. gondii, but displayed positive LAMP detection with P. vivax. The positive detection rate of the LAMP assay with PF3D7_1253300-5 was 90% (27/30), higher than that (80%, 24/30) of the positive rate of PF3D7_0112300 (18S rRNA) in examining clinical samples of P. falciparum infection cases. The LAMP assay with the primer set PF3D7_1253300-5 was more sensitive, specific, and accurate than those with PF3D7_0112300 (18S rRNA) in examining P. falciparum infection, and therefore it is a

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.

Multiplicity and molecular epidemiology of Plasmodium vivax and Plasmodium falciparum infections in East Africa.

PubMed

Zhong, Daibin; Lo, Eugenia; Wang, Xiaoming; Yewhalaw, Delenasaw; Zhou, Guofa; Atieli, Harrysone E; Githeko, Andrew; Hemming-Schroeder, Elizabeth; Lee, Ming-Chieh; Afrane, Yaw; Yan, Guiyun

2018-05-02

Parasite genetic diversity and multiplicity of infection (MOI) affect clinical outcomes, response to drug treatment and naturally-acquired or vaccine-induced immunity. Traditional methods often underestimate the frequency and diversity of multiclonal infections due to technical sensitivity and specificity. Next-generation sequencing techniques provide a novel opportunity to study complexity of parasite populations and molecular epidemiology. Symptomatic and asymptomatic Plasmodium vivax samples were collected from health centres/hospitals and schools, respectively, from 2011 to 2015 in Ethiopia. Similarly, both symptomatic and asymptomatic Plasmodium falciparum samples were collected, respectively, from hospitals and schools in 2005 and 2015 in Kenya. Finger-pricked blood samples were collected and dried on filter paper. Long amplicon (> 400 bp) deep sequencing of merozoite surface protein 1 (msp1) gene was conducted to determine multiplicity and molecular epidemiology of P. vivax and P. falciparum infections. The results were compared with those based on short amplicon (117 bp) deep sequencing. A total of 139 P. vivax and 222 P. falciparum samples were pyro-sequenced for pvmsp1 and pfmsp1, yielding a total of 21 P. vivax and 99 P. falciparum predominant haplotypes. The average MOI for P. vivax and P. falciparum were 2.16 and 2.68, respectively, which were significantly higher than that of microsatellite markers and short amplicon (117 bp) deep sequencing. Multiclonal infections were detected in 62.2% of the samples for P. vivax and 74.8% of the samples for P. falciparum. Four out of the five subjects with recurrent P. vivax malaria were found to be a relapse 44-65 days after clearance of parasites. No difference was observed in MOI among P. vivax patients of different symptoms, ages and genders. Similar patterns were also observed in P. falciparum except for one study site in Kenyan lowland areas with significantly higher MOI. The study used a novel method

Evolution of Fseg/Cseg dimorphism in region III of the Plasmodium falciparum eba-175 gene.

PubMed

Yasukochi, Yoshiki; Naka, Izumi; Patarapotikul, Jintana; Hananantachai, Hathairad; Ohashi, Jun

2017-04-01

The 175-kDa erythrocyte binding antigen (EBA-175) of the malaria parasite Plasmodium falciparum is important for its invasion into human erythrocytes. The primary structure of eba-175 is divided into seven regions, namely I to VII. Region III contains highly divergent dimorphic segments, termed Fseg and Cseg. The allele frequencies of segmental dimorphism within a P. falciparum population have been extensively examined; however, the molecular evolution of segmental dimorphism is not well understood. A comprehensive comparison of nucleotide sequences among 32 P. falciparum eba-175 alleles identified in our previous study, two Plasmodium reichenowi, and one P. gaboni orthologous alleles obtained from the GenBank database was conducted to uncover the origin and evolutionary processes of segmental dimorphism in P. falciparum eba-175. In the eba-175 nucleotide sequence derived from a P. reichenowi CDC strain, both Fseg and Cseg were found in region III, which implies that the original eba-175 gene had both segments, and deletions of F- and C-segments generated Cseg and Fseg alleles, respectively. We also confirmed the presence of allele with Fseg and Cseg in another P. reichenowi strain (SY57), by re-mapping short reads obtained from the GenBank database. On the other hand, the segmental sequence of eba-175 ortholog in P. gaboni was quite diverged from those of the other species, suggesting that the original eba-175 dimorphism of P. falciparum can be traced back to the stem linage of P. falciparum and P. reichenowi. Our findings suggest that Fseg and Cseg alleles are derived from a single eba-175 allele containing both segments in the ancestral population of P. falciparum and P. reichenowi, and that the allelic dimorphism of eba-175 was shaped by the independent emergence of similar dimorphic lineage in different species that has never been observed in any evolutionary mode of allelic dimorphism at other loci in malaria genomes. Copyright © 2017 Elsevier B.V. All

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hyperreactive malarial splenomegaly is associated with low levels of antibodies against red blood cell and Plasmodium falciparum derived glycolipids in Yanomami Amerindians from Venezuela.

PubMed

Vivas, Livia; O'Dea, Kieran P; Noya, Oscar; Pabon, Rosalba; Magris, Magda; Botto, Carlos; Holder, Anthony A; Brown, K Neil

2008-03-01

The immunological basis of the aberrant immune response in hyperreactive malarial splenomegaly (HMS) is poorly understood, but believed to be associated with polyclonal B cell activation by an unidentified malaria mitogen, leading to unregulated immunoglobulin and autoantibody production. HMS has been previously reported in Yanomami communities in the Upper Orinoco region of the Venezuelan Amazon. To investigate a possible association between antibody responses against Plasmodium falciparum and uninfected red blood cell (URBC) glycolipids and splenomegaly, a direct comparison of the parasite versus host anti-glycolipid antibody responses was made in an isolated community of this area. The anti-P. falciparum glycolipid (Pfglp) response was IgG3 dominated, whereas the uninfected red blood cell glycolipid (URBCglp) response showed a predominance of IgG1. The levels of IgG1 against Pfglp, and of IgG4 and IgM against URBCglp were significantly higher in women, while the anti-Pfglp or URBCglp IgM levels were inversely correlated with the degree of splenomegaly. Overall, these results suggest differential regulation of anti-parasite and autoreactive responses and that these responses may be linked to the development and evolution of HMS in this population exposed to endemic malaria. The high mortality rates associated with HMS point out that its early diagnosis together with the implementation of malaria control measures in these isolated Amerindian communities are a priority.

Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoforms PH1 and PH2 perturb vacuolar physiology.

PubMed

Callaghan, Paul S; Siriwardana, Amila; Hassett, Matthew R; Roepe, Paul D

2016-03-31

Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT). The approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter. During analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions. These data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.

Plasmodium falciparum Infection Status among Children with Schistosoma in Sub-Saharan Africa: A Systematic Review and Meta-analysis

PubMed Central

Degarege, Abraham; Degarege, Dawit; Veledar, Emir; Erko, Berhanu; Nacher, Mathieu; Beck-Sague, Consuelo M.; Madhivanan, Purnima

2016-01-01

Background It has been suggested that Schistosoma infection may be associated with Plasmodium falciparum infection or related reduction in haemoglobin level, but the nature of this interaction remains unclear. This systematic review synthesized evidence on the relationship of S. haematobium or S. mansoni infection with the occurrence of P. falciparum malaria, Plasmodium density and related reduction in haemoglobin level among children in sub-Saharan Africa (SSA). Methodology/Principal findings A systematic review in according with PRISMA guidelines was conducted. All published articles available in PubMed, Embase, Cochrane library and CINAHL databases before May 20, 2015 were searched without any limits. Two reviewers independently screened, reviewed and assessed all the studies. Cochrane Q and Moran’s I2 were used to assess heterogeneity and the Egger test was used to examine publication bias. The summary odds ratio (OR), summary regression co-efficient (β) and 95% confidence intervals (CI) were estimated using a random-effects model. Out of 2,920 citations screened, 12 articles (five cross-sectional, seven prospective cohort) were eligible to be included in the systematic review and 11 in the meta-analysis. The 12 studies involved 9,337 children in eight SSA countries. Eight studies compared the odds of asymptomatic/uncomplicated P. falciparum infection, two studies compared the incidence of uncomplicated P. falciparum infection, six studies compared P. falciparum density and four studies compared mean haemoglobin level between children infected and uninfected with S. haematobium or S. mansoni. Summary estimates of the eight studies based on 6,018 children showed a higher odds of asymptomatic/uncomplicated P. falciparum infection in children infected with S. mansoni or S. haematobium compared to those uninfected with Schistosoma (summary OR: 1.82; 95%CI: 1.41, 2.35; I2: 52.3%). The increase in odds of asymptomatic/uncomplicated P. falciparum infection among

Biosynthesis of GDP-fucose and Other Sugar Nucleotides in the Blood Stages of Plasmodium falciparum*

PubMed Central

Sanz, Sílvia; Bandini, Giulia; Ospina, Diego; Bernabeu, Maria; Mariño, Karina; Fernández-Becerra, Carmen; Izquierdo, Luis

2013-01-01

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions. PMID:23615908

Biosynthesis of GDP-fucose and other sugar nucleotides in the blood stages of Plasmodium falciparum.

PubMed

Sanz, Sílvia; Bandini, Giulia; Ospina, Diego; Bernabeu, Maria; Mariño, Karina; Fernández-Becerra, Carmen; Izquierdo, Luis

2013-06-07

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions.

Genome-level determination of Plasmodium falciparum blood-stage targets of malarial clinical immunity in the Peruvian Amazon.

PubMed

Torres, Katherine J; Castrillon, Carlos E; Moss, Eli L; Saito, Mayuko; Tenorio, Roy; Molina, Douglas M; Davies, Huw; Neafsey, Daniel E; Felgner, Philip; Vinetz, Joseph M; Gamboa, Dionicia

2015-04-15

Persons with blood-stage Plasmodium falciparum parasitemia in the absence of symptoms are considered to be clinically immune. We hypothesized that asymptomatic subjects with P. falciparum parasitemia would differentially recognize a subset of P. falciparum proteins on a genomic scale. Compared with symptomatic subjects, sera from clinically immune, asymptomatically infected individuals differentially recognized 51 P. falciparum proteins, including the established vaccine candidate PfMSP1. Novel, hitherto unstudied hypothetical proteins and other proteins not previously recognized as potential vaccine candidates were also differentially recognized. Genes encoding the proteins differentially recognized by the Peruvian clinically immune individuals exhibited a significant enrichment of nonsynonymous nucleotide variation, an observation consistent with these genes undergoing immune selection. A limited set of P. falciparum protein antigens was associated with the development of naturally acquired clinical immunity in the low-transmission setting of the Peruvian Amazon. These results imply that, even in a low-transmission setting, an asexual blood-stage vaccine designed to reduce clinical malaria symptoms will likely need to contain large numbers of often-polymorphic proteins, a finding at odds with many current efforts in the design of vaccines against asexual blood-stage P. falciparum. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Effect of naturally occurring Wolbachia in Anopheles gambiae s.l. mosquitoes from Mali on Plasmodium falciparum malaria transmission

PubMed Central

Gomes, Fabio M.; Hixson, Bretta L.; Tyner, Miles D. W.; Ramirez, Jose Luis; Canepa, Gaspar E.; Alves e Silva, Thiago Luiz; Molina-Cruz, Alvaro; Keita, Moussa; Kane, Fouseyni; Traoré, Boïssé; Sogoba, Nafomon; Barillas-Mury, Carolina

2017-01-01

A naturally occurring Wolbachia strain (wAnga-Mali) was identified in mosquitoes of the Anopheles gambiae complex collected in the Malian villages of Dangassa and Kenieroba. Phylogenetic analysis of the nucleotide sequence of two 16S rRNA regions showed that wAnga-Mali clusters with Wolbachia strains from supergroup A and has the highest homology to a Wolbachia strain isolated from cat fleas (Ctenocephalides). wAnga-Mali is different from two Wolbachia strains previously reported in A. gambiae from Burkina Faso (wAnga_VK5_STP and wAnga_VK5_3.1a). Quantitative analysis of Wolbachia and Plasmodium sporozoite infection in field-collected mosquitoes indicates that the prevalence and intensity of Plasmodium falciparum sporozoite infection is significantly lower in Wolbachia-infected females. The presence of Wolbachia in females from a laboratory Anopheles coluzzii (A. gambiae, M form) colony experimentally infected with P. falciparum (NF54 strain) gametocyte cultures slightly enhanced oocyst infection. However, Wolbachia infection significantly reduced the prevalence and intensity of sporozoite infection, as observed in the field. This indicates that wAnga-Mali infection does not limit early stages of Plasmodium infection in the mosquito, but it has a strong deleterious effect on sporozoites and reduces malaria transmission. PMID:29114059

Computational identification of signalling pathways in Plasmodium falciparum.

PubMed

Oyelade, Jelili; Ewejobi, Itunu; Brors, Benedikt; Eils, Roland; Adebiyi, Ezekiel

2011-06-01

Malaria is one of the world's most common and serious diseases causing death of about 3 million people each year. Its most severe occurrence is caused by the protozoan Plasmodium falciparum. Reports have shown that the resistance of the parasite to existing drugs is increasing. Therefore, there is a huge and urgent need to discover and validate new drug or vaccine targets to enable the development of new treatments for malaria. The ability to discover these drug or vaccine targets can only be enhanced from our deep understanding of the detailed biology of the parasite, for example how cells function and how proteins organize into modules such as metabolic, regulatory and signal transduction pathways. It has been noted that the knowledge of signalling transduction pathways in Plasmodium is fundamental to aid the design of new strategies against malaria. This work uses a linear-time algorithm for finding paths in a network under modified biologically motivated constraints. We predicted several important signalling transduction pathways in Plasmodium falciparum. We have predicted a viable signalling pathway characterized in terms of the genes responsible that may be the PfPKB pathway recently elucidated in Plasmodium falciparum. We obtained from the FIKK family, a signal transduction pathway that ends up on a chloroquine resistance marker protein, which indicates that interference with FIKK proteins might reverse Plasmodium falciparum from resistant to sensitive phenotype. We also proposed a hypothesis that showed the FIKK proteins in this pathway as enabling the resistance parasite to have a mechanism for releasing chloroquine (via an efflux process). Furthermore, we also predicted a signalling pathway that may have been responsible for signalling the start of the invasion process of Red Blood Cell (RBC) by the merozoites. It has been noted that the understanding of this pathway will give insight into the parasite virulence and will facilitate rational vaccine design

Comparison of Clinical Profile between P. vivax and P. falciparum Malaria in Children: A Tertiary Care Centre Perspective from India.

PubMed

Goyal, Jagdish Prasad; Makwana, Aarti M

2014-01-01

Background. Malaria is a one of the leading causes of morbidity and mortality in tropical countries. Plasmodium vivax (P. vivax) is usually thought to be causing benign malaria with low incidence of complications as compared to Plasmodium falciparum (P. falciparum). Methods. This retrospective observational study included malaria patients who were admitted to K.T. Children Hospital and P.D.U. Government Medical College, Rajkot, a tertiary care teaching hospital, Gujarat, western India, during the period January 2012 to December 2012. Inclusion criteria were patients in whom either P. falciparum or P. vivax was positive on rapid malaria antigen test and peripheral blood smear. Patients showing mixed infections were excluded from study. Results. A total of 79 subjects (mean age 5.4 ± 3.6 years) were included in the study. It consisted of 47 P. vivax and 32 P. falciparum cases. The P. vivax cases consisted of 33 (70.2%) males and 11 (19.8%) females while P. falciparum cases consisted of 14 (43.8%) males and 18 (56.2%) females. One patient of each P. vivax and P. falciparum expired. There was no statistical significant difference found between complications such as anemia, thrombocytopenia, liver and renal dysfunction, ARDS, and cerebral malaria between P. vivax and P. falciparum. Conclusion. We conclude that P. vivax monoinfection tends to have as similar course and complications as compared to malaria due to P. falciparum monoinfection.

Unusual Transmission of Plasmodium falciparum, Bordeaux, France, 2009

PubMed Central

Vareil, Marc-Olivier; Tandonnet, Olivier; Chemoul, Audrey; Bogreau, Hervé; Saint-Léger, Mélanie; Micheau, Maguy; Millet, Pascal; Koeck, Jean-Louis; Boyer, Alexandre; Rogier, Christophe

2011-01-01

Plasmodium falciparum malaria is usually transmitted by mosquitoes. We report 2 cases in France transmitted by other modes: occupational blood exposure and blood transfusion. Even where malaria is not endemic, it should be considered as a cause of unexplained acute fever. PMID:21291597

Streamlined, PCR-based testing for pfhrp2- and pfhrp3-negative Plasmodium falciparum.

PubMed

Parr, Jonathan B; Anderson, Olivia; Juliano, Jonathan J; Meshnick, Steven R

2018-04-02

Rapid diagnostic tests (RDTs) that detect histidine-rich protein 2 (PfHRP2) are used throughout Africa for the diagnosis of Plasmodium falciparum malaria. However, recent reports indicate that parasites lacking the pfhrp2 and/or histidine-rich protein 3 (pfhrp3) genes, which produce antigens detected by these RDTs, are common in select regions of South America, Asia, and Africa. Proving the absence of a gene is challenging, and multiple PCR assays targeting these genes have been described. A detailed characterization and comparison of published assays is needed to facilitate robust and streamlined testing approaches. Among six pfhrp2 and pfhrp3 PCR assays tested, the lower limit of detection ranged from 0.01 pg/µL to 0.1 ng/µL of P. falciparum 3D7 strain DNA, or approximately 0.4-4000 parasite genomes/µL. By lowering the elongation temperature to 60 °C, a tenfold improvement in the limit of detection and/or darker bands for all exon 1 targets and for the first-round reaction of a single exon 2 target was achieved. Additionally, assays targeting exon 1 of either gene yielded spurious amplification of the paralogous gene. Using these data, an optimized testing algorithm for the detection of pfhrp2- and pfhrp3-negative P. falciparum is proposed. Surveillance of pfhrp2- and pfhrp3-negative P. falciparum requires careful laboratory workflows. PCR-based testing methods coupled with microscopy and/or antigen testing serve as useful tools to support policy development. Standardized approaches to the detection of pfhrp2- and pfhrp3-negative P. falciparum should inform efforts to define the impact of these parasites.