scholarly journals Comparison of a SYBR Green I-Based Assay with a Histidine-Rich Protein II Enzyme-Linked Immunosorbent Assay for In Vitro Antimalarial Drug Efficacy Testing and Application to Clinical Isolates

2007 ◽  
Vol 51 (4) ◽  
pp. 1172-1178 ◽  
Author(s):  
David J. Bacon ◽  
Christine Latour ◽  
Carmen Lucas ◽  
Olga Colina ◽  
Pascal Ringwald ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Immunosorbent Assay ◽  
Sybr Green ◽  
Sybr Green I ◽  
Clinical Isolates ◽  
Enzyme Linked Immunosorbent Assay ◽  
Complete Medium ◽  
Phenol Red ◽  
Reference Strains

ABSTRACT In vitro drug susceptibility testing with the malaria parasite has been used to assess the antimalarial activities of new compounds and to monitor drug resistance in field isolates. We investigated the validity of a SYBR green I fluorescent-based assay under various culture conditions and compared the assay results to those of previously published histidine-rich protein II (HRPII) enzyme-linked immunosorbent assay (ELISA) methods. Reference strains of Plasmodium falciparum were cultured in vitro by using standard conditions in complete medium with and without phenol red before they were dispensed into 96-well plates predosed with chloroquine, mefloquine, or quinine. Following incubation, the culture supernatants were divided and the 50% inhibitory concentrations (IC50s) were determined by using a SYBR green I-based method and the HRPII capture ELISA method. There were no significant differences in IC50 values when phenol red was included in the medium. The IC50s and the IC90s of the antimalarials tested by both methods were similar or identical for each of the reference strains. Fresh clinical isolates of P. falciparum collected from imported cases of malaria in Lyon, France, were tested for in vitro resistance to chloroquine and mefloquine by using the validated SYBR green I and HRPII ELISA methods. The SYBR green I-based method was able to calculate IC50 and IC90 values similar or identical to those calculated by the HRPII assay with fresh clinical samples without removal of white blood cells. The SYBR green I-based method for determination of drug sensitivity levels produced results comparable to those produced by other methods, showing that this method can be used routinely to conduct surveillance for drug resistance in P. falciparum with fresh or cultured parasites.

scholarly journals Assessment and Continued Validation of the Malaria SYBR Green I-Based Fluorescence Assay for Use in Malaria Drug Screening

2007 ◽  
Vol 51 (6) ◽  
pp. 1926-1933 ◽  
Author(s):  
Jacob D. Johnson ◽  
Richard A. Dennull ◽  
Lucia Gerena ◽  
Miriam Lopez-Sanchez ◽  
Norma E. Roncal ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Culture Condition ◽  
Microtiter Plate ◽  
Growth Conditions ◽  
Drug Susceptibility ◽  
Sybr Green ◽  
Sybr Green I ◽  
Phenotypic Correlation ◽  
In Vitro Susceptibility

ABSTRACT Several new fluorescence malaria in vitro drug susceptibility microtiter plate assays that detect the presence of malarial DNA in infected erythrocytes have recently been reported, in contrast to traditional isotopic screens that involve radioactive substrate incorporation to measure in vitro malaria growth inhibition. We have assessed and further characterized the malaria SYBR Green I-based fluorescence (MSF) assay for its ability to monitor drug resistance. In order to use the MSF assay as a drug screen, all assay conditions must be thoroughly examined. In this study we expanded upon the capabilities of this assay by including antibiotics and antifolates in the drug panel and testing folic acid-free growth conditions. To do this, we evaluated a more expansive panel of antimalarials in combination with various drug assay culture conditions commonly used in drug sensitivity screening for their activity against Plasmodium falciparum strains D6 and W2. The detection and quantitation limits of the MSF assay were 0.04 to 0.08% and ∼0.5% parasitemia, respectively. The MSF assay quality was significantly robust, displaying a Z′ range of 0.73 to 0.95. The 50% inhibitory concentrations for each drug and culture condition combination were determined by using the MSF assay. Compared to the standard [3H]hypoxanthine assay, the MSF assay displayed the expected parasite drug resistance patterns with a high degree of global and phenotypic correlation (r 2 ≥ 0.9238), regardless of which culture condition combination was used. In conclusion, the MSF assay allows for reliable one-plate high-throughput, automated malaria in vitro susceptibility testing without the expense, time consumption, and hazard of other screening assays.

scholarly journals A Novel Oxazolidinone, Contezolid (MRX-I), Expresses Anti-Mycobacterium abscessus Activity In Vitro

2021 ◽  
Author(s):  
Qi Guo ◽  
Liyun Xu ◽  
Fusheng Tan ◽  
Yongjie Zhang ◽  
Junsheng Fan ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Abscessus ◽  
Clinical Isolates ◽  
Novel Approach ◽  
Reference Strains

An evaluation of the anti- M. abscessus activity expressed by a novel oxazolidinone, contezolid (MRX-I), toward 12 reference strains and 194 clinical isolates was conducted. Contezolid was active against M. abscessus in vitro , and comparable to the anti- M. abscessus effects of linezolid both extracellularly and intracellularly. Contezolid did not antagonize the most frequently used anti- M. abscessus drugs nor did pre-exposure to contezolid induce drug resistance. These results provide a novel approach to treating M. abscessus infections.

scholarly journals In Vitro Activity of Mirincamycin (U24729A) against Plasmodium falciparum Isolates from Gabon

2009 ◽  
Vol 54 (1) ◽  
pp. 540-542 ◽  
Author(s):  
Jana Held ◽  
Richard Westerman ◽  
Peter G. Kremsner ◽  
Benjamin Mordmüller
Keyword(s):  
Plasmodium Falciparum ◽  
Immunosorbent Assay ◽  
Incubation Time ◽  
Clinical Development ◽  
Vitro Activity ◽  
Clinical Isolates ◽  
Enzyme Linked Immunosorbent Assay ◽  
In Vitro Activity ◽  
Prolonged Incubation

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

scholarly journals Unreliable Measurement of Basophil Maximum Leukotriene Release with the Bühlmann CAST 2000 Enzyme-Linked Immunosorbent Assay Kit

2006 ◽  
Vol 13 (3) ◽  
pp. 420-422 ◽  
Author(s):  
S. E. Burastero ◽  
C. Paolucci ◽  
D. Breda ◽  
G. Monasterolo ◽  
R. E. Rossi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
False Positive ◽  
Positive Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Positive Results

ABSTRACT The Bühlmann CAST 2000 enzyme-linked immunosorbent assay is a potentially useful assay for measuring sulfidoleukotrienes released in vitro by allergen-challenged basophils. However, we observed that the positive-control reagent yielded positive signals in cell-free systems. These false-positive results depended on using a mouse anti-FcεRI monoclonal antibody and were prevented by degranulation-inducing reagents other than mouse monoclonal antibodies.

scholarly journals Adaptación y optimización de un método de lectura por fluorometría en el modelo farmacológico in vitro de cultivo de Plasmodium falciparum

2016 ◽  
Vol 45 (1) ◽  
pp. 127-146 ◽  
Author(s):  
María Helena Arias Marciales ◽  
Yinneth Victoria Rodríguez Novoa ◽  
Giovanny Garavito Cárdenas
Keyword(s):  
Plasmodium Falciparum ◽  
Sybr Green ◽  
Sybr Green I

<p>El modelo farmacológico de cultivo in vitro de P. falciparum es crucial en el tamizaje inicial de sustancias o extractos de plantas con posible actividad antiplasmodial. La densidad parasitaria puede determinarse mediante variadas metodologías, sin embargo, se han descrito numerosas ventajas y desventajas asociadas a cada una de ellas. Se evaluaron el tiempo de incubación necesario para la tinción y el uso de cultivos asincrónicos o sincrónicos en busca de valores óptimos; evidenciando un tiempo óptimo de 2 h, y límites de detección y cuantificación, menores en cultivos asincrónicos. Empleando las cepas FCR3 y FCB2 se evidenció un ruido de fondo de 12% y 38% respectivamente; la linealidad mostró una buena correlación, r2 de 0,9644 (FCR3) y 0,9841 (FCB2) y una pendiente de 1761,8 y 852,4 respectivamente. Además, se comprobó había concordancia entre los métodos, fluorométrico con SYBR Green I (SYBRG I) y microscópico con Giemsa, con diferencia media de 0,00002% y 0,09109% para FCR3 y FCB2 respectivamente. Los límites de detección y cuantificación fueron 0,5% y 1,5% de parasitemia. El factor Z con FCB2 fue 0,376, en tanto que con FCR3 alcanzó 0,702. La concentración inhibitoria 50 (CI50) frente a P. falciparum FCR3, generada por cloroquina (CQ) fue 0,37 mcg/mL por microscopía y 0,35 mcg/mL por fluorometría. Nuestros hallazgos sugieren que el ensayo de fluorescencia con SYBRG I, empleando fluorómetros comúnmente disponibles en muchos laboratorios, es preciso, robusto, rápido y exacto; para la evaluación in vitro de sustancias o extractos con posible actividad antiplasmodial.</p>

scholarly journals Desnutrição neonatal e produção de IFN-γ IL-12 e IL-10 por macrófagos/linfócitos: estudo da infecção celular, in vitro, por Staphylococcus aureus meticilina sensível e meticilina resistente

2012 ◽  
Vol 25 (5) ◽  
pp. 607-619 ◽  
Author(s):  
Thacianna Barreto da Costa ◽  
Natália Gomes de Morais ◽  
Thays Miranda de Almeida ◽  
Maiara Santos Severo ◽  
Célia Maria Machado Barbosa de Castro
Keyword(s):  
Staphylococcus Aureus ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ifn Γ

OBJETIVO: Avaliar a influência da desnutrição neonatal sobre a produção de Interferon gama, Interleucina-12 e Interleucina-10 em cultura de macrófagos alveolares e linfócitos infectados, in vitro, com Staphylococcus aureus sensível/resistente à meticilina. MÉTODOS: Ratos machos Wistar foram amamentados por mães cuja dieta, durante a lactação, continha 17% de proteína no grupo nutrido e 8% no grupo desnutrido. Após desmame, ambos os grupos receberam a dieta normoproteica. Os macrófagos foram obtidos após traqueostomia, através da coleta do lavado broncoalveolar. Para obtenção dos linfócitos, foi realizado o procedimento cirúrgico de punção cardíaca. Após o isolamento dos diferentes tipos celulares, procedeuse à realização dos estímulos com as cepas de estudo. A dosagem das citocinas foi realizada pelo método de Enzyme-Linked Immunosorbent Assay, a partir de amostras coletadas do sobrenadante das culturas após 24 horas de incubação. RESULTADOS: A desnutrição acarretou diminuição do crescimento ponderal, redução na produção de Interferon gama em cultura de macrófagos alveolares e linfócitos e diminuição na produção de Interleucina-12 em cultura de macrófagos alveolares. Apenas a produção de Interferon gama e Interleucina-10 em cultura de macrófagos alveolares apresentou diferença entre as cepas analisadas, em ambos os grupos estudados. CONCLUSÃO: O modelo de desnutrição neonatal produziu sequela no peso corporal e reduziu a produção de citocinas próinflamatórias (Interleucina-12 e Interferon gama), indicando que esse modelo de desnutrição pode comprometer a resolução de um processo infeccioso. A cepa de Staphylococcus aureus resistente à meticilina estimulou uma maior produção de Interferon gama e Interleucina-10 por macrófagos alveolares, o que sugeriu estimulação imunológica mais intensa, por essa cepa, nesse tipo celular especificamente.

Sign in / Sign up

Export Citation Format

Share Document