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1.
PLoS Negl Trop Dis ; 13(12): e0007885, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790397

RESUMO

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity.


Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Testes de Sensibilidade Parasitária/métodos , Animais , Células Cultivadas , Feminino , Humanos , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/parasitologia , Camundongos , Testes de Sensibilidade Parasitária/normas
2.
Parasit Vectors ; 12(1): 493, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640761

RESUMO

BACKGROUND: Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel. METHODS: Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72 h to measure viability as a function of NAD+/NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis. RESULTS: For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72 h, concordance remained a robust 74%. Further, robust Pearson's correlations (0.48-0.68) were measured between the assays. Of those actives at 72 h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline. CONCLUSIONS: The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified .


Assuntos
Descoberta de Drogas/métodos , Testes de Sensibilidade Parasitária/métodos , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose mansoni/tratamento farmacológico , Esquistossomicidas/farmacologia , Animais , Biomphalaria/parasitologia , Cricetinae , Feminino , Larva/classificação , Larva/efeitos dos fármacos , Estágios do Ciclo de Vida , Mesocricetus , Testes de Sensibilidade Parasitária/normas , Fenótipo , Schistosoma mansoni/classificação , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomicidas/uso terapêutico
3.
Parasitology ; 145(4): 453-463, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-27866478

RESUMO

For decades antimonials were the drugs of choice for the treatment of visceral leishmaniasis (VL), but the recent emergence of resistance has made them redundant as first-line therapy in the endemic VL region in the Indian subcontinent. The application of other drugs has been limited due to adverse effects, perceived high cost, need for parenteral administration and increasing rate of treatment failures. Liposomal amphotericin B (AmB) and miltefosine (MIL) have been positioned as the effective first-line treatments; however, the number of monotherapy MIL-failures has increased after a decade of use. Since no validated molecular resistance markers are yet available, monitoring and surveillance of changes in drug sensitivity and resistance still depends on standard phenotypic in vitro promastigote or amastigote susceptibility assays. Clinical isolates displaying defined MIL- or AmB-resistance are still fairly scarce and fundamental and applied research on resistance mechanisms and dynamics remains largely dependent on laboratory-generated drug resistant strains. This review addresses the various challenges associated with drug susceptibility and -resistance monitoring in VL, with particular emphasis on the choice of strains, susceptibility model selection and standardization of procedures with specific read-out parameters and well-defined threshold criteria. The latter are essential to support surveillance systems and safeguard the limited number of currently available antileishmanial drugs.


Assuntos
Antiprotozoários/efeitos adversos , Resistência a Múltiplos Medicamentos , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Testes de Sensibilidade Parasitária/normas , Anfotericina B/administração & dosagem , Anfotericina B/efeitos adversos , Anfotericina B/uso terapêutico , Animais , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Humanos , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Antimoniato de Meglumina/efeitos adversos , Antimoniato de Meglumina/uso terapêutico , Testes de Sensibilidade Parasitária/métodos , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Psychodidae/parasitologia , Recidiva
4.
Vet Parasitol ; 219: 84-99, 2016 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-27351036

RESUMO

This guideline is intended to assist in the planning and execution of studies designed to assess the efficacy of ectoparasiticides for fish. It is the first ectoparasite-specific guideline to deal with studies set in the aquatic environment and therefore provides details for the maintenance of environmental standards for finfish. Information is included on a range of pre-clinical study designs as well as clinical studies in commercial/production sites, set within a regulatory framework. It provides information on the study animals, their welfare, husbandry and environmental requirements during the study. The most commonly pathogenic ectoparasites are presented with relevant points regarding life history, host challenge and numeric evaluation. Preparation and presentation of both topical and oral test treatments is provided, together with guidance on data collection and analysis. The guideline provides a quality standard or efficacy studies on finfish, which will assist researchers and regulatory authorities worldwide and contribute to the wider objective of harmonisation of procedures.


Assuntos
Aquicultura/métodos , Testes de Sensibilidade Parasitária/métodos , Testes de Sensibilidade Parasitária/normas , Projetos de Pesquisa , Animais , Antiparasitários/farmacologia , Antiparasitários/uso terapêutico , Ectoparasitoses/tratamento farmacológico , Ectoparasitoses/veterinária , Doenças dos Peixes/tratamento farmacológico , Peixes , Parasitos/efeitos dos fármacos , Drogas Veterinárias/farmacologia
5.
Parasitol Res ; 112(3): 917-32, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23392903

RESUMO

Chemical disinfection is common practice and inevitable to achieve sufficient control over parasites particularly in intensive animal housing systems. To identify suitable chemicals, reliable data on antiparasitic efficacy of disinfectants are required. This review summarizes recently published experience with procedures applied to evaluate the viability of a variety of endoparasites following physical or chemical stress. It is concluded that laboratory models used to assess antiparasitic efficacy of e.g. commercial disinfectants should consider the most resistant stages of both helminths and protozoa, i.e. ascarid eggs and coccidia oocysts. To ensure reproducibility and transparency, standardized protocols are pivotal. Such protocols are established on a national level (e.g. DVG guidelines in Germany); however, internationally accepted certification procedures are currently lacking.


Assuntos
Antiparasitários/administração & dosagem , Desinfetantes/administração & dosagem , Desinfecção/métodos , Parasitos/efeitos dos fármacos , Animais , Alemanha , Guias como Assunto , Testes de Sensibilidade Parasitária/métodos , Testes de Sensibilidade Parasitária/normas
6.
Malar J ; 11: 325, 2012 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-22974086

RESUMO

BACKGROUND: Apparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values. METHODS: Performance parameters of the W2 P. falciparum clone (considered artemisinin "sensitive") were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC50s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS) and dihydroartemisinin (DHA) were verified by LC-MS analysis. Plasmodium falciparum field isolate IC50s for DHA from subjects in an artemisinin-resistant area in Cambodia were compared with W2 susceptibility. RESULTS: Plate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC50s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC50s for DHA for Cambodian field isolates were higher (p <0.0001) than the W2 clone (3.9 nM), both for subjects with expected (less than 72 hours; 6.3 nM) and prolonged (greater or equal to 72 hours; 9.6 nM) parasite clearance times during treatment with artesunate monotherapy. CONCLUSION: The W2 reference clone improved the interpretability of field isolate susceptibility from the immediate ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.


Assuntos
Antígenos de Protozoários/análise , Antimaláricos/farmacologia , Malária Falciparum/parasitologia , Testes de Sensibilidade Parasitária/métodos , Testes de Sensibilidade Parasitária/normas , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/análise , Artemisininas/farmacologia , Artesunato , Cromatografia Líquida , Meios de Cultura/química , Humanos , Concentração Inibidora 50 , Espectrometria de Massas , Plasmodium falciparum/isolamento & purificação
7.
J Ethnopharmacol ; 139(2): 678, 2012 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-22120015

RESUMO

In the literature circulates a classification system for antimicrobial activity, which has no basis whatsoever. In this commentary the non-existence of this classification system will be clearly indicated.


Assuntos
Anti-Infecciosos/uso terapêutico , Antiprotozoários/uso terapêutico , Descoberta de Drogas , Etnofarmacologia/normas , Testes de Sensibilidade Microbiana/normas , Testes de Sensibilidade Parasitária/normas , Fitoterapia/normas , Extratos Vegetais/uso terapêutico , Plantas Medicinais , Humanos
8.
Antimicrob Agents Chemother ; 56(2): 1105-7, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22123687

RESUMO

We have analyzed the in vitro activities of pyronaridine and methylene blue against 59 Plasmodium falciparum isolates from Kenya in association with polymorphisms in Pfcrt (codon 76), Pfmdr1 (codon 86), and Pfnhe (full sequence). The median inhibitory concentrations that kill 50% of parasites were 13.5 and 3.3 nM for pyronaridine and methylene blue, respectively. Their activities were not associated with polymorphisms in these genes. The drugs' high in vitro activities indicate that they would be efficacious against Kenyan isolates in vivo.


Assuntos
Antimaláricos/farmacologia , Malária Falciparum/parasitologia , Azul de Metileno/farmacologia , Naftiridinas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Humanos , Quênia , Testes de Sensibilidade Parasitária/normas , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/isolamento & purificação , Polimorfismo Genético , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
10.
J Antimicrob Chemother ; 66(3): 560-3, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21393228

RESUMO

OBJECTIVES: The aims of this study were to provide a cost-effective and valuable method for evaluating drug efficacy against Cryptosporidium parvum using a quantitative SYBR Green real-time PCR (qPCR) and to assess the efficacy of adenosine analogues as drug templates. METHODS: C. parvum HNJ-1 strain growing in human ileocaecal adenocarcinoma cells was employed as an in vitro culture system. To normalize the DNA extraction efficiency, a specific plasmid was added to each sample before DNA purification; the genomic DNA of infected cells was quantified by qPCR using specific primers to confirm drug efficacy and cytotoxicity. To determine the mechanism of action, enzymatic inhibition analyses were conducted using C. parvum S-adenosyl-l-homocysteine hydrolase (CpSAHH) recombinant protein. RESULTS: The dose-dependent growth inhibition of C. parvum was confirmed; 50% effective concentrations of neplanocin A (NPA) and 2-fluoroadenosine (2FA) were 139 µM and 0.842 µM, respectively. Cytotoxicity evaluation showed that the 50% growth inhibition concentration of 2FA was 1.18 µM; NPA did not exhibit any cytotoxicity up to 200 µM. The screening system revealed the specific but marginal efficacy of NPA and showed 2FA to be cytotoxic. Recombinant CpSAHH inhibition analyses showed that NPA competitively inhibited CpSAHH activity (K(i )= 0.395 µM), whereas 2FA did not. CONCLUSIONS: This novel qPCR system confirmed not only drug efficacy against C. parvum but also cytotoxicity to host cells. Moreover, since the SYBR Green method is cost effective, it could therefore be used in a wide variety of clinical and research-oriented applications of Cryptosporidium analysis.


Assuntos
Adenosina/análogos & derivados , Adenosina/farmacologia , Antiprotozoários/farmacologia , Cryptosporidium parvum/efeitos dos fármacos , DNA de Protozoário/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Adenosina/toxicidade , Antiprotozoários/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cryptosporidium parvum/crescimento & desenvolvimento , Cryptosporidium parvum/patogenicidade , DNA de Protozoário/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Humanos , Compostos Orgânicos/metabolismo , Testes de Sensibilidade Parasitária/métodos , Testes de Sensibilidade Parasitária/normas , Reação em Cadeia da Polimerase/normas , Coloração e Rotulagem/métodos
11.
Malar J ; 9: 375, 2010 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-21184684

RESUMO

BACKGROUND: The Worldwide Antimalarial Resistance Network (WWARN) is a global collaboration to support the objective that anyone affected by malaria receives effective and safe drug treatment. The Pharmacology module aims to inform optimal anti-malarial drug selection. There is an urgent need to define the drug exposure - effect relationship for most anti-malarial drugs. Few anti-malarials have had their therapeutic blood concentration levels defined. One of the main challenges in assessing safety and efficacy data in relation to drug concentrations is the comparability of data generated from different laboratories. To explain differences in anti-malarial pharmacokinetics in studies with different measurement laboratories it is necessary to confirm the accuracy of the assay methods. This requires the establishment of an external quality assurance process to assure results that can be compared. This paper describes this process. METHODS: The pharmacology module of WWARN has established a quality assurance/quality control (QA/QC) programme consisting of two separate components:1. A proficiency testing programme where blank human plasma spiked with certified reference material (CRM) in different concentrations is sent out to participating bioanalytical laboratories.2. A certified reference standard programme where accurately weighed amounts of certified anti-malarial reference standards, metabolites, and internal standards are sent to participating bioanalytical and in vitro laboratories. CONCLUSION: The proficiency testing programme is designed as a cooperative effort to help participating laboratories assess their ability to carry out drug analysis, resolve any potential problem areas and to improve their results - and, in so doing, to improve the quality of anti-malarial pharmacokinetic data published and shared with WWARN.By utilizing the same source of standards for all laboratories, it is possible to minimize bias arising from poor quality reference standards. By providing anti-malarial drug standards from a central point, it is possible to lower the cost of these standards.


Assuntos
Antimaláricos/farmacologia , Testes de Sensibilidade Parasitária/normas , Garantia da Qualidade dos Cuidados de Saúde/métodos , Padrões de Referência , Humanos , Cooperação Internacional , Malária/tratamento farmacológico , Malária/parasitologia , Testes de Sensibilidade Parasitária/métodos , Plasmodium/efeitos dos fármacos , Plasmodium/isolamento & purificação
12.
Parasitol Res ; 105(3): 825-34, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19452165

RESUMO

The ability to reliably detect anthelmintic resistance is a crucial part of resistance management. If data between countries are to be compared, the same test should give the same results in each laboratory. As the egg hatch test for benzimidazole resistance is used for both research and surveys, the ability of different laboratories to obtain similar results was studied through testing of known isolates of cyathostomins, Haemonchus contortus, Ostertagia ostertagi, and Cooperia oncophora in programs supported by the EU (Cost B16 and FP6-PARASOL). Initial results showed difficulties in obtaining reproducible and similar data within and between laboratories. A series of ring tests, i.e., simultaneous and coordinated rounds of testing of nematode isolates in different laboratories was subsequently performed. By adopting identical protocols, especially the use of deionized water and making dilutions of thiabendazole in dimethyl sulfoxide in the final ring test, laboratories correctly identified both susceptible and resistant isolates. The protocols for the test and preparation of solutions of thiabendazole are described.


Assuntos
Anti-Helmínticos/farmacologia , Benzimidazóis/farmacologia , Resistência a Medicamentos , Nematoides/efeitos dos fármacos , Testes de Sensibilidade Parasitária/métodos , Testes de Sensibilidade Parasitária/normas , Animais , Ovos , Haemonchus/efeitos dos fármacos , Ostertagia/efeitos dos fármacos , Reprodutibilidade dos Testes , Trichostrongyloidea/efeitos dos fármacos
13.
N Z Vet J ; 56(2): 55-9, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18408790

RESUMO

AIM: To evaluate the likely reliability of laboratory case submissions in assessing the prevalence of anthelmintic resistance in sheep nematodes in New Zealand, and to examine the possible influence of two alternative faecal nematode egg count reduction (FECR) analysis methodologies on such data. METHODS: A comparison was made between the prevalence of anthelmintic resistance determined using faecal egg count reduction tests (FECRTs) conducted on randomly selected sheep farms in a national survey with those derived from similar case material submitted to a veterinary pathology laboratory on a more ad-hoc basis. A comparison was also made between two alternative FECR analysis methodologies using the latter data. One methodology involved a partially differentiated procedure in which FECRs for individual nematode genera were only undertaken in those instances where reductions in total strongylid faecal nematode egg counts (FECs) (excluding Nematodirus) of <95% were recorded. The other was a fully differentiated method where reductions in FECs for individual parasites were undertaken in all cases. RESULTS: Although there were some differences between them the results showed that there were considerable similarities between the prevalence data obtained from both the national survey and laboratory case submissions. This was particularly evident in relation to the overall pattern of involvement of the various nematode genera and the types of anthelmintic concerned. A comparison between laboratory case submission data analysed using a partially differentiated FECR methodology with that of a fully differentiated procedure, however, suggested that the use of the former practice was likely to lead to the 'true' prevalence of resistance being underestimated. CONCLUSIONS: The results of this study indicate that examination of FECRT case submissions to veterinary laboratories may offer a useful source of information regarding changes in the prevalence of anthelmintic-resistant sheep nematodes in New Zealand. They also lend support to suggestions that the recently completed national survey may have provided a conservative estimate of the prevalence of such resistance.


Assuntos
Anti-Helmínticos/uso terapêutico , Resistência a Medicamentos , Helmintíase Animal/epidemiologia , Laboratórios/normas , Doenças dos Ovinos/epidemiologia , Medicina Veterinária , Animais , Fezes/parasitologia , Helmintíase Animal/tratamento farmacológico , Helmintíase Animal/etiologia , Helmintíase Animal/parasitologia , Laboratórios/estatística & dados numéricos , Nova Zelândia/epidemiologia , Avaliação de Resultados em Cuidados de Saúde , Contagem de Ovos de Parasitas/veterinária , Testes de Sensibilidade Parasitária/normas , Testes de Sensibilidade Parasitária/veterinária , Prevalência , Reprodutibilidade dos Testes , Ovinos , Doenças dos Ovinos/tratamento farmacológico , Doenças dos Ovinos/etiologia , Doenças dos Ovinos/parasitologia
14.
Antimicrob Agents Chemother ; 52(1): 288-98, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17954693

RESUMO

Implemented as one arm of the malaria control program in French Guiana in the early 1990s, our laboratory has since established in vitro profiles for parasite drug susceptibility to a panel of eight antimalarials for more than 1,000 Plasmodium falciparum isolates from infected patients. The quinine-doxycycline combination was introduced in 1995 as the first-line drug treatment against uncomplicated P. falciparum malaria, replacing chloroquine, and the first-line drug combination was changed to the artemether-lumefantrine combination in 2002. Resistance to chloroquine declined 5 years after it was dropped in 1995 as the first-line drug, but unlike similar situations in Africa, there was a rapid halt to this decline. Doxycycline susceptibility substantially decreased from 2002 to 2005, suggesting parasite selection under quinine-doxycycline drug pressure. Susceptibility to mefloquine decreased from 1997 onward. Throughout the period from 1994 to 2005, most isolates were sensitive in vitro to quinine, amodiaquine, and atovaquone. Susceptibility to amodiaquine was strongly correlated with that to chloroquine and to a lesser extent with that to mefloquine and halofantrine. Susceptibilities to mefloquine and to halofantrine were also strongly correlated. There were two alerts issued for in vitro artemether resistance in the period from 2002 to 2003 and again in 2005, both of which could be associated with the presence of an S769N polymorphism in the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-type P. falciparum ATPase6 (PfATPase6) gene. Analysis of susceptibility to lumefantrine, conducted for the first time in 2005, indicates an alarming rate of elevated 50% inhibitory concentrations. In vitro monitoring of parasite drug susceptibility should be pursued to further document the consequences of specific drug policies on the local parasite population and, in particular, to establish profiles of susceptibility to individual components of drug combinations to provide early warning signs of emerging parasite resistance.


Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/classificação , Antimaláricos/uso terapêutico , Resistência a Medicamentos/genética , Guiana Francesa/epidemiologia , Genótipo , Política de Saúde , Humanos , Malária Falciparum/parasitologia , Testes de Sensibilidade Parasitária/normas , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética
15.
N Z Vet J ; 54(6): 365-6, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17151740

RESUMO

In a recent communication (McKenna 2006), a comparison was made between four different methods for calculating results from faecal egg count reduction (FECR) tests (FECRTs). The first and most complex of these, referred to as FECRT1, involved the use of the formula: FECR = 100 x (1-[T2/T1][C1/C2]), where T1 and T2 represented the mean pre- and post-treatment faecal nematode egg counts (FECs) of a treated group, and C1 and C2 represented the mean pre- and post-treatment FECs of an untreated control group, respectively. The other three formulae consisted of more simplified versions of this procedure. In one of them (FECRT2), only post-treatment samples were considered, whereas the other two were based on comparisons between the FECs of groups of animals sampled at the time of anthelmintic treatment (pre-treatment) with those sampled several days later (post-treatment). Thus, FECRT2 was determined according to the formula: FECR = 100 x (1-[T2/C2]), while FECRT3 was calculated from FECR = 100 x (1-[T2/T1]). The fourth procedure (FECRT4) was based on a further simplification of FECRT3, where pre-treatment FECs from only one treatment group were used for comparison with all post-treatment results. This base-line pre-treatment group thus effectively functioned as an untreated control group and hence the formula for FECRT4 was FECR = 100 x (1-[T2/C1]). The study was based on an analysis of 210 previously published FECRTs performed in sheep or goats. In each case, FECRs were calculated using all four of these FECRT formulae, and their results compared. The results of these comparisons indicated that the use of any one of them was likely to result in similar estimates of anthelmintic efficacy and the detection of comparable numbers of cases of anthelmintic resistance continued.


Assuntos
Anti-Helmínticos/farmacologia , Resistência a Medicamentos , Fezes/parasitologia , Contagem de Ovos de Parasitas/veterinária , Testes de Sensibilidade Parasitária/veterinária , Animais , Testes de Sensibilidade Parasitária/normas , Sensibilidade e Especificidade
16.
Am J Trop Med Hyg ; 75(5): 777-82, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17123965

RESUMO

Correlation studies on the in vitro drug response of field isolates of Plasmodium falciparum and molecular markers for drug resistance are becoming important as many malaria control programs abandon monotherapies and resort to combination therapies. The standardization and optimization of the in vitro drug sensitivity assay are one of the prerequisites for validating molecular markers in the field. The present study was designed to assess and compare the growth of freshly obtained isolates for at least the first erythrocytic cycle in various culture media and determine the in vitro response to chloroquine in alternative media. Parasite growth was consistently higher in Dulbecco's modified Eagle's medium (DME)-human serum, Iscove's modified Dulbecco's medium (IMDM)-human serum, RPMI 1640 medium-goat serum, and a serum-free medium containing 1:1 (v/v) mixture of IMDM and F-12 supplemented with an ammonium sulfate fraction of adult bovine serum than in RPMI 1640 medium-human serum mixture. The level of chloroquine response determined in human serum-supplemented DME, IMDM, and RPMI 1640 media did not differ significantly (P > 0.05) from the control (RPMI 1640-human serum). This study suggests that alternative media may be used to optimize parasite growth during the critical initial phase of transition from in vivo to in vitro conditions. The capacity of these media to support long-term cultivation of P. falciparum requires further investigation.


Assuntos
Cloroquina/farmacologia , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Testes de Sensibilidade Parasitária/normas , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Animais , Camarões/epidemiologia , Meios de Cultura/química , Epidemiologia Molecular , Plasmodium falciparum/crescimento & desenvolvimento , Albumina Sérica
17.
J Ethnopharmacol ; 106(3): 290-302, 2006 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-16698208

RESUMO

Natural products, either as pure compounds or as standardized plant extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity. To secure this, a number of pivotal quality standards need to be set at the level of extract processing and primary evaluation in pharmacological screening models. This review provides a number of recommendations that will help to define a more sound 'proof-of-concept' for antibacterial, antifungal, antiviral and antiparasitic potential in natural products. An integrated panel of pathogens is proposed for antimicrobial profiling, using accessible standard in vitro experimental procedures, endpoint parameters and efficacy criteria. Primary requirements include: (1) use of reference strains or fully characterized clinical isolates, (2) in vitro models on the whole organism and if possible cell-based, (3) evaluation of selectivity by parallel cytotoxicity testing and/or integrated profiling against unrelated micro-organisms, (4) adequately broad dose range, enabling dose-response curves, (5) stringent endpoint criteria with IC(50)-values generally below 100microug/ml for extracts and below 25microM for pure compounds, (6) proper preparation, storage and in-test processing of extracts, (7) inclusion of appropriate controls in each in vitro test replicate (blanks, infected and reference controls) and (8) follow-up of in vitro activity ('hit'-status) in matching animal models ('lead'-status).


Assuntos
Anti-Infecciosos/uso terapêutico , Antiprotozoários/uso terapêutico , Testes de Sensibilidade Microbiana/normas , Testes de Sensibilidade Parasitária/normas , Fitoterapia/normas , Extratos Vegetais/uso terapêutico , Plantas Medicinais , Humanos , Guias de Prática Clínica como Assunto
18.
Am J Trop Med Hyg ; 69(2): 168-73, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-14506772

RESUMO

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.


Assuntos
Amodiaquina/análogos & derivados , Antimaláricos/farmacologia , Meios de Cultura , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Testes de Sensibilidade Parasitária/normas , Plasmodium falciparum/efeitos dos fármacos , Amodiaquina/farmacologia , Animais , Camarões/epidemiologia , Cloroquina/farmacologia , Humanos , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Pirimetamina/farmacologia , Albumina Sérica
19.
J Clin Microbiol ; 41(7): 2992-3000, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12843032

RESUMO

Six multipurpose contact lens solutions [All-in-One, All-in-One (Light), ReNu MultiPlus, Optifree Express, Complete, and Solo-care soft] were tested for their efficacies against Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique for amoebic enumeration. Against trophozoites, All-in-One, ReNu Multiplus, and Optifree Express achieved total kill (log reduction of >3) after the manufacturer's minimum recommended disinfection time (MMRDT), with the remaining solutions failing to reach a log reduction of 1. After 24 h of exposure, all solutions proved trophozoiticidal, achieving, with the exception of Complete (log reduction of 3.13), total kill. Against cysts, All-in-One gave a log reduction of >3 within the MMRDT, with all other solutions failing to achieve a log reduction of 1. After 24 h of exposure, All-in-One achieved total kill of cysts (log reduction of 3.74), ReNu MultiPlus gave a log reduction of 3.15, and the remaining solutions reached log reductions of between 1.09 and 2.27. The MPN technique provides a simple, reliable, and reproducible method of amoebic enumeration that depends on simply establishing the presence or absence of growth on culture plates inoculated with a series of dilutions and determining the MPN of amoebae present from statistical tables. By use of this technique, two of the multipurpose solutions tested, ReNu MultiPlus and Optifree Express, demonstrated effective trophozoiticidal activities within the recommended disinfection times; however, only All-in-One proved effective against both trophozoites and cysts over the same time period. This MPN technique, which uses axenically produced trophozoites and mature, double-walled cysts, has the potential to form the basis of a national standard for amoebicidal efficacy testing of multipurpose contact lens disinfecting solutions.


Assuntos
Acanthamoeba/efeitos dos fármacos , Soluções para Lentes de Contato/farmacologia , Acanthamoeba/crescimento & desenvolvimento , Acanthamoeba/isolamento & purificação , Animais , Contagem de Colônia Microbiana , Meios de Cultura , Desinfecção/métodos , Testes de Sensibilidade Parasitária/métodos , Testes de Sensibilidade Parasitária/normas , Reprodutibilidade dos Testes , Fatores de Tempo
20.
Biomedica ; 22(2): 211-8, 2002 Jun.
Artigo em Espanhol | MEDLINE | ID: mdl-12152485

RESUMO

The WHO method for determining insecticide resistance was standardized for several species of Lutzomyia sand flies under laboratory and field conditions. The biological assays were applied solely to optimize the conditions for the control, i.e., without insecticide, and to estimate mortality due to handling or other unfavorable conditions. Adult female flies from 3 laboratory colonies and one field strain were tested: two laboratory strains of Lutzomyia longipalpis, one laboratory strain of Lutzomyia serrana and one field-collected strain of Lutzomyia quasitownsendi. The WHO method was compared with one modified in which, during the post-exposure period, the recommended plain tube apparatus was replaced with a plastic container layered with damp plaster of Paris. Three paper substrate types were compared under each condition: olive oil additive, silicon oil additive and plain paper. The measured variable was percent mortality in 24 h. For the WHO protocol, the L. longipalpis strains indicated a 0-10% mortality, L. serrana 20-80% and L. quasitownsendi 10-50%. With the modified WHO apparatus, the average mortality was < 4% for all species. No significant differences were observed among the paper treatments. These results indicate a strong species-specific effect of post-exposure conditions on sand flies. To establish baseline levels of insecticide resistance in Lutzomyia sand flies, the WHO method is recommended only for L. longipalpis, and the modified method for L. serrana, L. quasitownsendi and closely related species.


Assuntos
Inseticidas , Testes de Sensibilidade Parasitária/normas , Psychodidae , Organização Mundial da Saúde , Animais , Feminino , Leishmaniose/parasitologia , Masculino , Padrões de Referência , Especificidade da Espécie
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