261 research outputs found
Assessment and monitoring of antimalarial drug efficacy for the treatment of uncomplicated falciparum malaria
No full textContributors: Peter Bloland, Centers for Disease Control and Prevention, Atlanta, GA, United States of America; Pascal Ringwald, Roll Back Malaria/Partnership Secretariat, World Health Organization, Geneva, Switzerland; Robert W. Snow, University of Oxford, United Kingdom; Kenya Medical Research Institute, Nairobi, KenyaWHO/HTM/RBM/2003.50who2003_monitoring.pd
Episodic absorption in the outflow of V603 Aquilae
No full textWe report on the time-dependent behaviour of ultraviolet spectral lines in Hubble Space Telescope Goddard High-Resolution Spectrograph data of the classical nova V603 Aql. In particular, episodic blueshifted absorption (extending to ∼−2500 km s−1) is present, with a variability time-scale down to ∼1 min. The data provide a rare opportunity to study the rapid evolution of absorption structures that may be associated with accretion-disc winds in cataclysmic variables. At least three absorption events are recorded (at blueward velocities only) over ∼5 h, each lasting ∼10–15 min. The derived velocity, acceleration and optical depth properties provide an empirical picture of stochastically variable structures in the outflow, with no evidence for short-term (less than ∼1 h) cyclic or modulated behaviour in the overall absorption properties. In contrast, the emission components of the ultraviolet resonance lines are very stable in velocity and strength in this low-inclination system. On at least two occasions there is an intriguing short-term ‘flare’ in the ultraviolet continuum flux (of up to ∼40 per cent). Though there is no clear one-to-one relation in these data between the continuum fluctuations and the occurrence of the absorption events, the time-scales for the two variable phenomena are essentially the same. The irregular absorption episodes in the ultraviolet data of V603 Aql presently defy a clear physical interpretation. Their overall characteristics are discussed in the context of instabilities in radiation-pressure-driven disc winds
Conférence technique de l'OCEAC
No full textLa physiopathologie de l'accès pernicieux est extrêmement complexe et encore mal connue. La séquestration d'hématies parasitées et l'intervention de certaines cytokines sont vraisemblables, mais il reste encore à élucider pourquoi certains cas seulement évoluent vers une forme grave. Une meilleure compréhension de la physiopathologie pourrait permettre la mise au point de traitements adaptés pour une réduction significative de la létalité liée à ces formes sévères. (Résumé d'auteur
Conférence technique de l'OCEAC
No full textLa physiopathologie de l'accès pernicieux est extrêmement complexe et encore mal connue. La séquestration d'hématies parasitées et l'intervention de certaines cytokines sont vraisemblables, mais il reste encore à élucider pourquoi certains cas seulement évoluent vers une forme grave. Une meilleure compréhension de la physiopathologie pourrait permettre la mise au point de traitements adaptés pour une réduction significative de la létalité liée à ces formes sévères. (Résumé d'auteur
A database of antimalarial drug resistance
No full textA large investment is required to develop, license and deploy a new antimalarial drug. Too often, that investment has been rapidly devalued by the selection of parasite populations resistant to the
drug action. To understand the mechanisms of selection, detailed information on the patterns of drug use in a variety of environments, and the geographic and temporal patterns of resistance is needed. Currently, there is no publically-accessible central database that contains information on the levels of resistance to antimalaria drugs. This paper outlines the resources that are available and the steps that might be taken to create a dynamic, open access database that would include current and historical data on clinical efficacy, in vitro responses and molecular markers related to drug resistance in Plasmodium falciparum and
Plasmodium vivax. The goal is to include historical and current data on resistance to commonly used drugs, like chloroquine and sulfadoxine-pyrimethamine, and on the many combinations that are now being tested in different settings. The database will be accessible to all on the Web. The information in such a database will inform optimal utilization of current drugs and sustain the longest possible therapeutic life of newly introduced drugs and combinations. The database will protect the valuable investment represented by the development and deployment of novel
therapies for malaria.The study was supported by a grant to CHS from the US National Institutes of Health, AI 55604
Molecular epidemiology of malaria in Yaounde, Cameroon. I. Analysis of point mutations in the dihydrofolate reductase-thymidylate synthase gene of P[astnodiutn fakiparum.
No full textAbstract. Pyrimethamine and cycloguanil, the major human metabolite of proguanil, are inhibitors of dihydrofolate reductase that play a key role in the treatment and prevention of chloroquine-resistant Plasmodium fakiparum infections in sub-Saharan Africa. Resistance to these antifolate drugs has emerged in some areas of Africa. Earlier molecular studies have demonstrated that point mutations at key positions of the dihydrofolate reductase-thymidylate synthase gene are strongly associated with antifolate resistance. However, whether the same or distinct mutations are involved in the development of resistance to both pyrimethamine and cycloguanil has not been well established in naturally occurring P. fakiparum isolates. In this study, the in vitro responses to both antifolate drugs were measured in 42 Cameroonian isolates and compared with the complete sequence of the dihydrofolate reductase domain of the gene (from 34 of 42 isolates) to analyze the genotype that may distinguish between pyrimethamine and cycloguanil resistance. The wild-type profile (n = 11 isolates) was associated with low 50% inhibitory concentrations (IC,,,s) ranging from 0.32 to 21.4 nanamole for pyrimethamine and 0.60-6.40 nM for cycloguanil. Mutant isolates had at least one amino acid substitution, Asn-108. Only three mutant codons were observed among the antifolate-resistant isolates: Ue-51, Arg-59, and Asn-108. The increasing number of point mutations was associated with an increasing level of pyrimethamine IC,,, and, to a much lesser extent, cycloguanil IC5,,. These results support a partial crossresistance between pyrimethamine and cycloguanil that is based on similar amino acid substitutions in dihydrofolate reductase and suggest that two or three mutations, including at least Asn-108, may be necessary for cycloguanil resistance, whereas a single Asn-108 mutation is sufficient for pyrimethamine resistance. Pyrimethamine and proguanil are dihydrofolate reductase @HFR) inhibitors that continue to play a major role in the chemotherapy of chloroquine-resistant Plasniodiurn falcipar u m 1 Initially used as a monotherapy in the 1950s and 1960s, both of these DHFR inhibitors are currently used in combination with another antimalarial drug to delay the emergence of resistant strains and/or to enhance their specific activity against the malaria Sulfadoxinepyrimethamine is indicated for the treatment of acute, uncomplicated malarial attacks due to chloroquine-resistant P. falciparunz infections in areas where this drug combination remains generally effective, such as in most of the African continent. F'roguanil, in combination with chloroquine, is recommended for chemoprophylaxis in nonimmune individuals traveling to certain areas where malaria is endemic and in pregnant women residing in malaria-endemic zones? Proguanil is also used for the treatment of multidrug-resistant P. f a k i p a r u m infections in combination with a new antiprotozoan drug, ato~aquone.6*~ Pyrimethamine and cycloguanil, the biologically active human metabolite of proguanil, share similar chemical structures and inhibit the same molecular target.8-g These two features suggest a potential for cross-resistance between the drugs. However, earlier in vivo and in vitro studies have drawn contradictory conclusions? Another approach based on the analysis of the genetic mechanism of antifolate resistance may help resolve this question. Comparison of the P. falciparum dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene sequences from several reference clones has suggested genetic profìles that are associated with pyrimethamine resistance and cycloguanil resistance.10*' * According to these studies, a Ser-to-Asn substitution at position 108 confers resistance to pyrimethamine, while a Ser-to-Thr substitution at position 108, associated with a Ala-to-Val substitution at position 16, confers resistance to cycloguanil. Ancillary mutations at positions 51, 59, and/or 164 are associated with elevated levels of antifolate resistance. These molecular criteria for antifolate resistance have not been fully confirmed in naturally occurring isolates of P. fakiparum. In our previous studies on Cameroonian isolates, the key dhfr codon was compared with the phenotype defined by the in vitro sensitivity or resistance to pyrimethamine.I2 In the present study, the in vitro responses to both pyrimethamine and cycloguanil were determined for clinical isolates obtained in Cameroon and the DHFR domain of the dhfr-ts gene was fully sequenced, with the aim to establish whether there is a particular genotype that defines pyrimethamine and cycloguanil resistance. PATIENTS, MATERIALS, AND METHODS Patients. Most of the patients participated in the clinical trials conducted in Yaounde, Cameroon between 1996 and 1998. Inclusion criteria included an age 2 5 years old, fever at consultation (or history of fever within the past 24 hr), a monoinfection with P. falciparum based on the microscopic examination of Giemsa-stained thin and thick blood smears, a parasite density >5,000 asexual parasites/pl of blood; and no recent history of self-medication with antimalarial drugs, as confirmed by a negative Saker-Solomons urine test rest11t.I~ Patients with signs and symptoms of severe and complicated malaria, as defined by the World Health Organizawere excluded. Depending on the clinical conditions, the patients were keated with the first-line (amodiaquine), second-line (sulfadoxine-pyrimethamine), or third-line drug (quinine) used in Cameroon. Informed consent was obtained from the patient or the patient's guardian in the case of children. The study was approved by the Cameroonian National Ethics Committee and the Cameroonian Ministry of Public Health. Parasite DNA. Forty-two clinical isolates of P. falciparum were obtained by venipuncture before treatment. Venous blood samples (5-10 ml of whole blood) were collected in a tube coated with an anticoagulant (EDTA) (Vacutainer; Terumo Europe N V , Leuven, Belgium) and washed three times in folate-and p-aminobenzoic acid-free RPMI 1640 medium by centrifugation (2,000 X g for 10 min) within 3 hr after blood collection. An aliquot of 1.5-2 m l of the red blood cell pellet was used to extract parasite DNA (contam-' inated with human leukocyte DNA). Infected erythrocytes were suspended in 15 m l of ice-cold NET buffer (150 mM NaCl, 10 mM EDTA, 50 mM Tris, pH 7.5) and lysed with 0.015% saponin. The lysate was centrifuged at 2,000 X g for 10 min and the pellet was transferred to a 1.5-ml microfuge tube and suspended in 500 pl of NET buffer. The mixture was treated with 1% N-lauroylsarcosine (Sigma Chemical Co., St. Louis, MO) and RNAse A (100 pg/ml) at 37°C for 1 hr and proteinase K (200 pg/ m l ) at 50°C for 1 hr. unit of Pwo DNA polymerase (Roche Diagnostics, Meylan, France) in a 50-pI reaction at 94°C for 2 min for the first cycle and 30 sec in subsequent cycles, 50°C for 1 min for the first cycle and 30 sec in subsequent cycles, and 72°C for 1 min in all cycles, for a total of 30 cycles. The primers were designed on the basis of the complete P. falciparuin dhfr-ts seq~ence.'~J~ Five microliters of the amplification product were loaded on a 1.2% agarose gel, subjected to electrophoresis, stained with ethidium bromide, and visualized under ultraviolet transillumination to confirm the presence of the 708-basepair DNA fragment. Sequencing of DNA. One of the primers was phosphorylated at its 5'-end by incubating 1 nanomole of primer in a mixture containing 0.5 mM ATP, buffer (10 mM MgCl,, 5 mh4 dithiothreitol, 70 mM Tris-HCI, pH 7.6), and 20 units of T4 polynucleotide kinase (New England Biolabs, Inc., Beverly, MA), in a volume Of 100 p1 at 37°C for 15 min. The kinase was heat-inactivated at 65°C for 20 min. The 5'-end-labeled primer was used to perform the polymerase chain reaction described above. The amplified products were purified by glass beads (Jetsorb; Genomed Inc., Research 5'-ATGATGGAACAAGTCTGCGACGTTTTCGAT-3' i . Triangle Park, NC) and treated with lambda exonuclease (Gibco-BRL Life Technologies, Cergy Pontoise, France) to generate single-stranded DNA. The single-stranded template was used to sequence the amplified product by the dideoxy chain termination rea~ti0n.l~ According to the previous studies, 5 amino acids at positions 16, 51, 59, 108, and 164 of the dhfr-ts gene undergo mutational changes.lOJIJs The wild-type genotype, designated the 3D7-type profile, was defined as Ala-16/Asn-5 1ICys-59/Ser-108me-164. The mutant genotype with a single Serto-Asn-I08 mutation was designated the HB3-type profile. The mutant genotypes with a double mutation were designated the Kl-type (Asn-108/Arg-59), the 7G8-type (Asn108me-51), or the FCR3-type (Val-16/Thr-108). The mutant genotype with triple mutations Asn-108/Arg-59/Ile-5 1 was designated the WZ-type. The genotype with quadruple mutations Asn-10WArg-59me-5 1Leu-164 was designated the Cambodian type.19 In vitro assay. In vitro drug sensitivity assays were performed on the clinical isolates without prior adaptation to the in vitro culture conditions. Infected erythrocytes were suspended in the complete folate-and p-aminobenzoic acidfree RPMI 1640 medium consisting of 10% non-immune human serum, 25 mM HEPES, 25 mM NaHCO, at a hematocrit of 1.5% and an initial parasitemia of 0.2-1.0%. If the blood sample had a parasitemia >1.0%, fresh uninfected, type A+ erythrocytes were added to adjust the parasitemia to 0.6%. The isotopic microtest developed by Desjardins and otherszo was used in this study.2' Two hundred microliters of the suspension of infected erythrocytes were distributed in each well of 96-well tissue culture plates. The parasites were incubated at 37°C in 5% CO, for 18 hr. 3H-hypoxanthine (1 pCi/well; Amersham International, Plc., Buckinghamshire, United Kingdom) was added to assess parasite growth. After an additional 48 hr of incubation, the plates were frozen to terminate the in vitro drug sensitivity assay. The plates were thawed, and the contents of each well were collected on glass-fiber filter papers, washed, and dried using a cell harvester. The filter disks were transferred into scintillation tubes, and 2 m l of scintillation cocktail (Organic Counting Scintillant@; Amersham International, Plc.) were added. The incorporation of 3H-hypoxanthine was quantitated using a liquid scintillation counter (Wallac 1409; Pharmacia, Uppsala, Sweden). The 50% inhibitory concentration (IC,,), defined as the drug concentration corresponding to 50% of the uptake of 3H-hypoxanthine measured in the drug-free control wells, was determined by non-linear regression analysis of logarithm of concentrations plotted against the parasite growth inhibition. The best-fitting sigmoid curve was derived by using the Prismm software (Graphpad Software, Inc., San Diego, CA). The pyrimethamine IC,, values were classified as sensitive ((100 nM), moderately resistant (100-2,000 nM), and highly resistant (>ZOO0 nM), as in our previous s t~d i e s .~. '~ Similarly, the IC,, values for cycloguanil were classified as sensitive ( 6 0 nM), moderately resistant (50-500 nM), and highly resistant (>500 nM). These threshold values have been defined arbitrarily to separate three distinct in vitro responses observed in our earlier s t~~d i e s .~~, *~ More recently, Nzila-Mounda and others have also observed distinct groups of Kenyan isolates based on the IC,, of antifolate drugs.24 The validity of these cut-off values, in particular their possible relevance to in vivo responses, has not been established. Data were expressed as geometric mean IC,, and range. Correlation of the logarithmic values of IC5o for pyrimethamine and cycloguanil was calculated by a linear regression analysis. Data were analyzed by using the Statview software (Abacus Concepts, Inc., Calabasas, CA). RESULTS The in vitro activity of pyrimethamine and cycloguanil was determined for 42 clinical isolates. Using the earlier classification of in vitro responses to antifolate drugs, 18 isolates were pyrimethamine-sensitive (geometric mean IC50 = 4.01 nM, range = 0.32-84.8 nM) The in vitro responses to pyrimethamine and cycloguanil were highly correlated (r = 0.935, P < 0.05). The complete sequence of the DHFR domain of the dhfrts gene was determined in 34 isolates. Amino acid substitutions occurred at positions 51, 59, and 108. Eleven isolates with the wild-type (3D7-type) profile had geometric mean (range) pyrimethamine ICso and cycloguanil IC,, values of . ues of pyrimethamine (70.6 n M and 119 nM) and cycloguanil (8.1 nM and 25.8 nM) for these two HB3-type isolates were higher than the range of values for the wild-type isolates. The Arg-59/Asn-l08 double mutation (Kl-type profile) was found in two isolates with even higher IC,, values for both pyrimethamine (396 nM and 854 nM) and cycloguanil (56.5 n M and 78.7 nM). The triple mutation Ile-51/ Arg-59/Asn-108 (W2-type) was detected in 14 isolates presenting elevated ICso values for pyrimethamine and cycloguanil. The ICso values of five isolates with mixed alleles were within the range of values displayed by mutant isolates. None of the isolates had the mutant codons Val-16, Thr-108, and Leu-164. DISCUSSION As in the previous studies, the in vitro response of pyrimethamine and cycloguanil was highly c~r r e l a t e d .~~-~~ These results are in agreement with the fact that pyrimethamine and cycloguanil share similar chemical structures and inhibit the same e n~y m e .~.~ Eleven isolates were characterized by the wild-type dhfr profile and exhibited low IC,, values. These data allow us to deduce that the phenotype and wildtype genotype correspond to the antifolate-sensitive pattern. The presence of one (Asn-108) or two (Asn-108 + Arg-59) mutations in the dhfr-ts gene was clearly associated with an increasing level of pyrimethamine IC5o values. Although the mean pyrimethamine IC,, values for isolates with a triple mutation were higher than the IC,, values for isolates with a double mutation, the latter values were within the range of values observed in isolates with a triple mutation. Our data thus suggest a stepwise increment in the level of resistance to pyrimethamine that is directly related to the presence and number of point mutations, but there was no clearcut difference in the level of resistance between the isolates ~~ ~ n 274 BASCO AND RINGWALD d with double or triple mutations. AS for cycloguanil, the relationship between the HB3-type :ingle mutation and cycloguanil resistance was not evident from our data. However, the K1-type double mutations (Asn-108 + Arg-59) and the W2-type triple mutations (Asn-108 4-Arg-59 + showed some degree of association with an elevated IC,, for When the range of IC,, values for pyrimethamine is compared between the wild-type isolates (0.32-21.4 nM) and mutant isolates with one (71 and 119 nM) to three point mutations (323-1 1,700 nM), it can be deduced that the cutoff point for in vitro pyrimethamine resistance is between 22 and 70 nM. The cut-off value for in vitro cycloguanil resistance cannot be estimated in the present study due to the wide dispersion of the IC,, and the existence of some mutant isolates with IC,, values between 10 and 50 nM
Comparison of in vivo and in vitro tests of resistance in patients treated with chloroquine in Yaoundé, Cameroon
No full textLa résistance de #Plasmodium falciparum à la chloroquine a été décrite dans tous les pays de l'Afrique subsharienne. Néanmoins, la chloroquine reste le médicament de première intention pour le traitement de l'accès palustre simple dans la plupart des pays africains. L'extension de la résistance à la chloroquine nécessite une surveillance permanente soit par des tests in vivo, soit par des tests in vitro. Afin de rechercher une concordance entre ces deux types de test, nous avons comparé les résultats du nouveau test d'efficacité thérapeutique introduit par l'OMS en 1996 à ceux du semi-microtest isotopique. Ce nouveau test in vivo est basé sur l'évolution, après un traitement standard par la chloroquine à 25 mg/kg sur 3 jours, de l'état clinique et de la parasitémie chez des malades atteints d'un accès palustre simple à #P. falciparum. Les résultats sont exprimés en réponse clinique adéquate ou en échec thérapeutique précoce ou tardif en fonction de la disparition, de l'aggravation, de la persistance ou de la réapparition des signes cliniques, en particulier la fièvre, et en fonction de l'évolution de la parasitémie. Le semi-microtest consiste à étudier la croissance in vitro des parasites en présence de concentrations croissantes de chloroquine (25 à 1600 nmol/l). La croissance est mesurée par l'incorporation d'hyposanthine tritiée. Les résultats sont exprimés en concentration inhibitrice 50% (Cl90) correspondant à la concentration inhibant la croissance de 50% des parasites par rapport à un témoin. Le seuil de résistance pour la chloroquine est fixé à 100 nmol/l. Parmi les 117 malades inclus, 102 (87%) ont été suivis pendant 14 jours, et 95 tests in vitro réalisés avec les isolats des malades (46 enfants et 49 adultes) ont put être interprétés... (D'après résumé d'auteur
Molecular epidemiology of malaria in Yaoundé, Cameroon : 3. Analysis of chloroquine resistance and point mutations in the multidrug resistance 1 (pfmdr 1) gene of Plasmodium falciparum
No full textIt has been postulated that chloroquine resistance may be associated with a single point mutation at codon 86 of the #Plasmodium falciparum$ multidrug resistance 1 (pfmdr 1) gene. Using a simple and rapid molecular technique involving polymerase chain reaction and restriction fragment length polymorphism, the frequency of the Asn-to-Tyr mutation associated with chloroquine resistance was established among 129 clinical isolates obtained from indigenous patients in Yaoundé, Cameroon. The results showed that 110 of 129 isolates display a mutant codon. The other clinical isolates had either a pure wild-type Asn-86 codon (n = 12) or mixed Asn/Tyr alleles (n = 7). In vitro drug assays were performed to compare the genotype and phenotype in 102 clinical isolates. Of these isolates, 86 displayed pure Tyr-86 mutant codon ; 48 (56%) mutant isolates were chloroquine-resistant (50% inhibitory concentration [IC50] > 100 nM), as expected, but 38 (44%) mutant isolates were chloroquine-sensitive (IC50 < 100 nM). Three chloroquine-resistant isolates and seven chloroquine-sensitive parasites carried a wild-type Asn-86 codon. Mixed alleles were found in six isolates (four chloroquine-sensitive and two chloroquine-resistant isolates). Our results did not confirm previous observations on the possible association between chloroquine resistance phenotype and genotype based on the pfmdr 1 gene. (Résumé d'auteur
Chloroquine resistance in Plasmodium falciparum and polymorphism of the CG2 gene
No full textA distinct genotype (designated Dd2-type profile) consisting of 12 point mutations and 3 repetitive regions of the CG2 gene, a candidate gene for chloroquine resistance, has been associated with in vitro resistance in laboratory-adapted strains of #Plasmodium falciparum$. The DNA sequence of clinical isolates, characterized by in vitro and in vivo tests, was analyzed to evaluate whether the genotype corresponds to the phenotype in naturally occurring parasites. Eight of 11 chloroquine-resistant isolates had the Dd2 genotype. One resistant isolate (by in vitro assay) with a sensitive CG2 genotype was sensitive in vivo. Two resistant isolates and 6 sensitive isolates were multiple infections with mixed alleles. No typical CG2 genotype was found corresponding to the chloroquine-sensitive isolates. These results suggest a strong association between the drug-resistant and CG2 genotypes and support the hypothesis that the CG2 gene may be implicated in chloroquine resistance. (Résumé d'auteur
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