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>

In-vitro culture of Plasmodium falciparum: Utility of modified (RPNI) medium for drug-sensitivity studies using SYBR Green I assay

2011 ◽  
Vol 127 (1) ◽  
pp. 318-321 ◽  
Author(s):  
Shubhra Singh ◽  
Rajeev Kumar Srivastava ◽  
Mukesh Srivastava ◽  
S.K. Puri ◽  
K. Srivastava
Keyword(s):  
Plasmodium Falciparum ◽  
In Vitro Culture ◽  
Drug Sensitivity ◽  
Sybr Green ◽  
Sybr Green I ◽  
Sensitivity Studies

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

2013 ◽  
Vol 12 (1) ◽  
Author(s):  
Neils B Quashie ◽  
Nancy O Duah ◽  
Benjamin Abuaku ◽  
Lydia Quaye ◽  
Ruth Ayanful-Torgby ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Sybr Green ◽  
Clinical Isolates ◽  
In Vitro Test ◽  
Vitro Test

Plasmodium falciparum: Development and validation of a measure of intraerythrocytic growth using SYBR Green I in a flow cytometer

2009 ◽  
Vol 121 (2) ◽  
pp. 144-150 ◽  
Author(s):  
Shinji Izumiyama ◽  
Mako Omura ◽  
Tomohiko Takasaki ◽  
Hiroshi Ohmae ◽  
Hiroko Asahi
Keyword(s):  
Plasmodium Falciparum ◽  
Flow Cytometer ◽  
Sybr Green ◽  
Sybr Green I ◽  
Development And Validation

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 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 of Malaria In Vitro Drug Combination Screening and Mixed-Strain Infections Using the Malaria Sybr Green I-Based Fluorescence Assay

2009 ◽  
Vol 53 (6) ◽  
pp. 2557-2563 ◽  
Author(s):  
Edgie-Mark A. Co ◽  
Richard A. Dennull ◽  
Drew D. Reinbold ◽  
Norman C. Waters ◽  
Jacob D. Johnson
Keyword(s):  
Drug Combination ◽  
Antimalarial Drug ◽  
Fixed Ratio ◽  
Drug Combinations ◽  
Sybr Green ◽  
Sybr Green I ◽  
The Individual ◽  
Parasite Populations ◽  
Concentration Curves

ABSTRACT Several drug development strategies, including optimization of new antimalarial drug combinations, have been used to counter malaria drug resistance. We evaluated the malaria Sybr green I-based fluorescence (MSF) assay for its use in in vitro drug combination sensitivity assays. Drug combinations of previously published synergistic (atovaquone and proguanil), indifferent (chloroquine and azithromycin), and antagonistic (chloroquine and atovaquone) antimalarial drug interactions were tested against Plasmodium falciparum strains D6 and W2 using the MSF assay. Fifty percent inhibitory concentrations (IC50s) were calculated for individual drugs and in fixed ratio combinations relative to their individual IC50s. Subsequent isobologram analysis and fractional inhibitory concentration determinations demonstrated the expected drug interaction pattern for each combination tested. Furthermore, we explored the ability of the MSF assay to examine mixed parasite population dynamics, which are commonly seen in malaria patient isolates. Specifically, the capacity of the MSF assay to discern between single and mixed parasite populations was determined. To simulate mixed infections in vitro, fixed ratios of D6 and W2 strains were cocultured with antimalarial drugs and IC50s were determined using the MSF assay. Dichotomous concentration curves indicated that the sensitive and resistant parasites composing the genetically heterogeneous population were detectable. Biphasic analysis was performed to obtain subpopulation IC50s for comparison to those obtained for the individual malaria strains alone. In conclusion, the MSF assay allows for reliable antimalarial drug combination screening and provides an important method to discern between homogenous and heterogeneous parasite populations.

scholarly journals Flow Cytometric Enumeration of Parasitemia in Cultures ofPlasmodium falciparumStained with SYBR Green I and CD235A

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Jin Woo Jang ◽  
Ju Yeon Kim ◽  
Jung Yoon ◽  
Soo Young Yoon ◽  
Chi Hyun Cho ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Microscopic Examination ◽  
Blood Cells ◽  
Detection Method ◽  
Microscopic Analysis ◽  
Sybr Green ◽  
Sybr Green I ◽  
Facs Analysis ◽  
Clear Separation ◽  
Flow Cytometric

A flow cytometric (FACS) detection method forPlasmodium falciparumcultures (P. falciparum) was developed using SYBR Green I and CD235A and compared against the Giemsa stained microscopic examination. The culturedP. falciparumwere spiked into red blood cells (RBCs) to yield parasitemia, ranging from 0.01% to 22.0%. FACS analysis demonstrated a clear separation betweenP. falciparuminfected and uninfected RBCs. The measured percentage of parasitemia by FACS revealed higher precision (CV of 2.2–37.2%) with the sensitivity of 0.01% parasitemia than Giemsa stained microscopic examination (CV of 7.2–66.0%). High correlation of measured parasitaemia (r=0.98,P<0.05) was observed between FACS and Giemsa stained microscopic analyses. The higher levels of parasitaemia detection were observed in all ranges by FACS in comparison to Giemsa stained microscopic analysis. The currently reported FACS method using SYBR Green I and CD235A is potentially useful for measuring parasitemia in treating patients.

scholarly journals Development of New PCR Assay with SYBR Green I for Detection of Mycoplasma, Acholeplasma, and Ureaplasma sp. in Cell Cultures

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 876
Author(s):  
Jolanta Krzysztoń-Russjan ◽  
Jakub Chudziak ◽  
Małgorzata Bednarek ◽  
Elżbieta Lidia Anuszewska
Keyword(s):  
Cell Cultures ◽  
Limit Of Detection ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Time Saving ◽  
Detection And Identification ◽  
Polymerase Chain

Mycoplasma, Acholeplasma, and Ureaplasma sp. are atypical bacteria responsible for in vitro cell culture contaminations that can warp the results. These bacteria also cause human and animal infections and may lead to chronic diseases. In developed polymerase chain reaction (PCR) in this study a quantitative PCR with SYBR Green I fluorochrome was applied to facilitate the Mycoplasma, Acholeplasma, and Ureaplasma sp. DNA detection and identification. Screening Test-1 v.1 (triplex qPCR) allowed for the detection of 11 species. Test-1 v.2 (three single qPCRs) pre-identified three subgroups, allowing for the reduction of using single qPCRs in Test-2 for species identification. The range of both tests was consistent with pharmacopeial requirements for microbial quality control of mammal cells and included detection of M. arginini, M. orale, M. hyorhinis, M. fermentans, M. genitalium, M. hominis, M. pneumoniae, M. salivarium, M. pirum, A. laidlawii, and U. urealyticum. Limit of detection values varied between 125–300 and 50–100 number of copies per milliliter in Test-1 and Test-2, respectively. Test-1 and Test-2 showed fully concordant results, allowed for time-saving detection and/or identification of selected species from Mycoplasma, Acholeplasma, and Ureaplasma in tested cell cultures.

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