sybr-green-i and Parasitemia

Rapid and accurate tools are needed for high-throughput in vitro antibabesial drug testing. In this study, flow cytometry for the measuring of Babesia bovis in vitro culture, was developed using SYBR Green I and compared against the results of fluorescence-based assay and microscopic assay. A high correlation of measured parasitemia was observed with high R

Topics: Babesia bovis; Erythrocytes; Flow Cytometry; Humans; Parasitemia

Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations.. Plasmodium falciparum cultures were synchronized with single layer Percoll during the schizont stage of the intraerythrocytic development cycle. Cultures were left to reinvade to early ring-stage and parasitaemia was quantified using flow cytometry. Cultures were diluted to 2% haematocrit and 0.5% parasitaemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700 nM of dihydroartemisinin or 0.02% dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples.. 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of - 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date.. eRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.

Topics: Antimalarials; Artemisinins; Benzothiazoles; Diamines; Drug Resistance; Erythrocytes; Flow Cytometry; Fluorescent Dyes; Half-Life; Humans; Malaria, Falciparum; Organic Chemicals; Parasitemia; Plasmodium falciparum; Povidone; Quinolines; Real-Time Polymerase Chain Reaction; Silicon Dioxide

In epidemiological surveys and surveillance the application of molecular tools is essential in detecting submicroscopic malaria. A genus-specific conventional cytochrome b (cytb) PCR has shown high sensitivity in field studies, detecting 70% submicroscopic malaria. The main objective of this study was to assess the conversion from conventional to real-time PCR testing both SYBR and probe protocols, and including quantitative (q) PCR. The protocols were assessed applying well-defined clinical patient material consisting of 33 positive and 80 negative samples. Sequencing of positive PCR products was performed. In addition, a sensitivity comparison of real-time PCR methods was done by including five relevant assays investigating the effect of amplification target and platform. Sensitivity was further examined using field material consisting of 111 P.falciparum positive samples from Tanzanian children (< 5 years), as well as using related patient data to assess the application of q-PCR with focus on low-level parasitaemia. Both the cytb SYBR and probe PCR protocols showed as high sensitivity and specificity as their conventional counterpart, except missing one P. malariae sample. The SYBR protocol was more sensitive and specific than using probe. Overall, choice of amplification target applied is relevant for achieving ultra-sensitivity, and using intercalating fluorescence dye rather than labelled hydrolysis probes is favourable. Application of q-PCR analysis in field projects is important for the awareness and understanding of low-level parasitaemia. For use in clinical diagnosis and epidemiological studies the highly sensitive and user-friendly cytb SYBR q-PCR method is a relevant tool. The genus-specific method has the advantage that species identification by sequencing can be performed as an alternative to species-specific PCR.

Topics: Benzothiazoles; Child, Preschool; Cytochromes b; Diamines; DNA, Protozoan; Genes, Protozoan; Humans; Infant; Infant, Newborn; Malaria; Organic Chemicals; Parasitemia; Plasmodium falciparum; Quinolines; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Sensitivity and Specificity

The redox metabolism of the malaria parasite Plasmodium falciparum and its human host has been suggested to play a central role for parasite survival and clearance. A common approach to test hypotheses in redox research is to challenge or rescue cells with pro- and antioxidants. However, quantitative data on the susceptibility of infected erythrocytes towards standard redox agents is surprisingly scarce. Here we determined the IC

Topics: Acetylcysteine; Antioxidants; Ascorbic Acid; Benzothiazoles; Diamide; Diamines; Dithiothreitol; Dose-Response Relationship, Drug; Erythrocytes; Fluorescent Dyes; Host-Parasite Interactions; Humans; Hydrogen Peroxide; Inhibitory Concentration 50; Malaria, Falciparum; Organic Chemicals; Oxidants; Oxidation-Reduction; Oxidative Stress; Parasitemia; Plasmodium falciparum; Quinolines; tert-Butylhydroperoxide; Time Factors

The diagnosis of malaria, caused by Plasmodium spp., still remains a challenging process. Especially in low-income countries, a rapid user-friendly method is needed for the efficient care of the patient. A small-angle light scattering device consisting of hardware and software was developed. Using the DNA-binding dye SYBR Green, malaria infections could be distinguished in healthy red blood cells infected with Plasmodium. Subsequently, samples from parasite positive and negative patients living in a hyper-endemic area of Kinshasa, DRC were assessed. The scatter profiles were distinct and malaria infection could be detected using the Giemsa stain. Although these results are preliminary, they indicate that the device has the potential to be used as a new diagnostic tool for the detection of Malaria infection.

Topics: Azure Stains; Benzothiazoles; Diamines; Erythrocytes; Fluorescent Dyes; Humans; Light; Malaria, Falciparum; Organic Chemicals; Parasitemia; Plasmodium falciparum; Quinolines; Scattering, Radiation

The malaria SYBR green assay, which is used to profilein vitrodrug susceptibility ofPlasmodium falciparum, is a reliable drug screening and surveillance tool. Malaria field surveillance efforts provide isolates with various low levels of parasitemia. To be advantageous, malaria drug sensitivity assays should perform reproducibly among various starting parasitemia levels rather than at one fixed initial value. We examined the SYBR green assay standardized procedure developed by the Worldwide Antimalarial Resistance Network (WWARN) for its sensitivity and ability to accurately determine the drug concentration that inhibits parasite growth by 50% (IC50) in samples with a range of initial parasitemia levels. The initial sensitivity determination of the WWARN procedure yielded a detection limit of 0.019% parasitemia.P. falciparumlaboratory strains and field isolates with various levels of initial parasitemia were then subjected to a range of doses of common antimalarials. The IC50s were comparable for laboratory strains with between 0.0375% and 0.6% parasitemia and for field isolates with between 0.075% and 0.6% parasitemia for all drugs tested. Furthermore, assay quality (Z') analysis indicated that the WWARN procedure displays high robustness, allowing for drug testing of malaria field samples within the derived range of initial parasitemia. The use of the WWARN procedure should allow for the inclusion of more malaria field samples in malaria drug sensitivity screens that would have otherwise been excluded due to low initial parasitemia levels.

Topics: Antimalarials; Artemisinins; Atovaquone; Benzothiazoles; Biological Assay; Chloroquine; Diamines; DNA, Protozoan; Drug Resistance; Erythrocytes; Fluorescent Dyes; Humans; Inhibitory Concentration 50; Malaria, Falciparum; Mefloquine; Organic Chemicals; Parasitemia; Plasmodium falciparum; Public Health Surveillance; Quinolines; Reproducibility of Results; Sensitivity and Specificity

A flow cytometric (FACS) detection method for Plasmodium falciparum cultures (P. falciparum) was developed using SYBR Green I and CD235A and compared against the Giemsa stained microscopic examination. The cultured P. falciparum were spiked into red blood cells (RBCs) to yield parasitemia, ranging from 0.01% to 22.0%. FACS analysis demonstrated a clear separation between P. falciparum infected 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.

Topics: Azure Stains; Benzothiazoles; Diamines; Erythrocytes; Flow Cytometry; Glycophorins; Humans; Malaria, Falciparum; Microscopy; Organic Chemicals; Parasitemia; Plasmodium falciparum; Quinolines; Staining and Labeling

High-throughput methods for evaluation of in vivo efficacy of candidate compounds against Plasmodium parasites are necessary during the antimalarial drug development process. It is essential that enumeration of parasitemia in the infected blood from experimental host animals is accurate and reliable. Flow cytometric enumeration of parasitized cells stained with fluorescent dye is a rapid alternative method to conventional microscopic counting. In this study, a protocol for flow cytometric enumeration of rodent malaria parasite Plasmodium berghei-infected red blood cells (RBC) stained with SYBR Green I was developed. The optimal concentration of SYBR Green I used to stain infected RBC was 4× for 30 min. This SYBR Green I staining protocol in combination with the bi-dimensional FL-1(530)/FL-3(620) detection method accurately detects parasitemia above 0.02%. The dye is stable during the prolonged incubation period necessary for accurate enumeration of parasitemia, with no loss of fluorescent signal over a period of hours. This protocol was validated in an antimalarial assay and the result was comparable to that obtained from conventional microscopic counting. The SYBR Green I flow cytometric protocol is thus a rapid and precise tool for high-throughput in vivo antimalarial drug screening.

Topics: Animals; Antimalarials; Benzothiazoles; Diamines; Erythrocytes; Flow Cytometry; Fluorescent Dyes; Mice; Organic Chemicals; Parasitemia; Parasitology; Plasmodium berghei; Quinolines; Staining and Labeling

The erythrocytic life cycle of Plasmodium falciparum is highly associated with severe clinical symptoms of malaria that causes hundreds of thousands of death each year. The parasite develops within human erythrocytes leading to the disruption of the infected red blood cell (iRBC) prior to the start of a new cycle of erythrocyte infection. Emerging mechanisms of resistance against antimalarial drugs require improved knowledge about parasite's blood stages to facilitate new alternative antimalarial strategies. For the analysis of young blood stages of Plasmodium at the molecular level, the isolation of ring stages is essential. However, early stages can hardly be separated from both, late stages and non-infected red blood cells using conventional methods. Here, iRBCs were stained with the DNA-binding dyes Vybrant® DyeCycle™ Violet and SYBR® Green I. Subsequently, cells were subjected to flow-cytometric analysis. This enabled the discrimination of early stage iRBCs as well as late-stage iRBCs from non-infected erythrocytes and the properties of the used dyes were evaluated. Moreover, early stage iRBCs were isolated with high purity (>98%) by FACS. Subsequently, development of sorted early stages of the parasite was monitored over time and compared with control cultures. The described flow cytometry method, based on staining with Vybrant DyeCycle Violet, allows the isolation of viable ring stages of the malarial agent P. falciparum, and thereby provides the basis for new, broad-range molecular investigations of the parasite.

Topics: Azure Stains; Benzothiazoles; Diamines; Erythrocytes; Flow Cytometry; Fluorescent Dyes; Humans; Malaria; Organic Chemicals; Parasitemia; Plasmodium falciparum; Quinolines; Schizonts; Staining and Labeling; Time Factors

To compare the applicability of the SYBR Green-I assay with the standard schizont maturation assay, for determination of sensitivity of Plasmodium vivax (P. vivax) to chloroquine and a new antifolate WR 99210.. The study was conducted at Mae Tao Clinic for migrant workers, Tak Province during April 2009 to July 2010. A total of 64 blood samples (1 mL blood collected into sodium heparinized plastic tube) were collected from patients with mono-infection with P. vivax malaria prior to treatment with standard regimen of a 3-day chloroquine. In vitro sensitivity of P. vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-I assays.. A total of 30 out of 64 blood samples collected from patients with P. vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays. The failure rates of the schizont maturation inhibition assay (50%) and the SYBR Green-I assay (54%) were similar (P=0.51). The median IC10s, IC50s and IC90s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P. vivax tested. Based on the cut-off of 100 nM, the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates, respectively. The strength of agreement between the two methods was very poor for both chloroquine and WR99210.. On the basis of this condition and its superior sensitivity, the microscopic method appears better than the SYBR Green-I Green assay for assessing in vitro sensitivity of fresh P. vivax isolates to antimalarial drugs.

Topics: Antimalarials; Benzothiazoles; Chloroquine; Diamines; Humans; Inhibitory Concentration 50; Malaria, Vivax; Organic Chemicals; Parasitemia; Parasitic Sensitivity Tests; Plasmodium vivax; Quinolines; Schizonts

Variability in the ability of the malaria parasite Plasmodium falciparum to invade human erythrocytes is postulated to be an important determinant of disease severity. Both the parasite multiplication rate and erythrocyte selectivity are important parameters that underlie such variable invasion. We have established a flow cytometry-based method for simultaneously calculating both the parasitemia and the number of multiply-infected erythrocytes. Staining with the DNA-specific dye SYBR Green I allows quantitation of parasite invasion at the ring stage of parasite development. We discuss in vitro and in vivo applications and limitations of this method in relation to the study of parasite invasion.

Topics: Benzothiazoles; Diamines; Erythrocytes; Flow Cytometry; Fluorescent Dyes; Host-Parasite Interactions; Humans; Malaria, Falciparum; Microscopy; Organic Chemicals; Parasitemia; Plasmodium falciparum; Quinolines; Sensitivity and Specificity; Severity of Illness Index

Validation of the sensitivity of the SYBR Green I in vitro test against an enzyme-linked immunosorbent assay (ELISA)-based drug sensitivity assay. Our results suggest that the SYBR Green I assay is a fast and inexpensive malaria drug screening assay for laboratory use. However, because of its lack of sensitivity in whole blood samples its usefulness for testing clinical samples may be limited.

Topics: Animals; Antimalarials; Benzothiazoles; Diamines; Drug Resistance; Enzyme-Linked Immunosorbent Assay; Humans; Organic Chemicals; Parasitemia; Parasitic Sensitivity Tests; Plasmodium falciparum; Quinolines; Sensitivity and Specificity

Improvements on malarial diagnostic methods are currently needed for the correct detection in low-density Plasmodium falciparum infections. Microfluorimetric DNA-based assays have been previously used for evaluation of anti-malarial drug efficacy on Plasmodium infected erythrocytes. Several factors affecting the sensitivity of these methods have been evaluated, and tested for the detection and quantification of the parasite in low parasitaemia conditions.. Parasitaemia was assessed by measuring SYBRGreen I (SGI) and PicoGreen (PG) fluorescence of P. falciparum Dd2 cultures on human red blood cells. Different modifications of standard methods were tested to improve the detection sensitivity. Calculation of IC50 for chloroquine was used to validate the method.. Removal of haemoglobin from infected red-blood cells culture (IRBC) increased considerably the fluorescent signal obtained from both SGI and PG. Detergents used for cell lysis also showed to have an effect on the fluorescent signal. Upon depletion of haemoglobin and detergents the fluorescence emission of SGI and PG increased, respectively, 10- and 60-fold, extending notably the dynamic range of the assay. Under these conditions, a 20-fold higher PG vs. SGI fluorescent signal was observed. The estimated limits of detection and quantification for the PG haemoglobin/detergent-depleted method were 0.2% and 0.7% parasitaemia, respectively, which allow the detection of ~10 parasites per microliter. The method was validated on whole blood-infected samples, displaying similar results as those obtained using IRBC. Removal of white-blood cells prior to the assay allowed to increase the accuracy of the measurement, by reducing the relative uncertainty at the limit of detection from 0.5 to 0.1.. The use of PG microassays on detergent-free, haemoglobin-depleted samples appears as the best choice both for the detection of Plasmodium in low-density infections and anti-malarial drugs tests.

Topics: Animals; Benzothiazoles; Cell Separation; Chloroquine; Cytophotometry; Diamines; DNA, Protozoan; Erythrocytes; Fluorescent Dyes; Hemoglobins; Humans; Inhibitory Concentration 50; Limit of Detection; Malaria, Falciparum; Organic Chemicals; Parasitemia; Parasitic Sensitivity Tests; Plasmodium falciparum; Quinolines; Sensitivity and Specificity; Staining and Labeling

For the diagnosis of imported malaria, optical or immunochromatographic methods are known to be less sensitive and less specific than PCR-based methods, which are conversely more complicated and time-consuming. An original strategy, based upon the sequential use of a multiplex competitive real-time PCR detecting Plasmodium falciparum or Plasmodium spp. infection, followed by, if necessary, a single real-time PCR for species identification, was therefore performed and then tested versus conventional PCR in routine conditions. Conventional PCR has been used since October 1999 in the Department of Parasitology, University Hospitals in Toulouse, as a 2nd line diagnostic method. Out of 183 patients tested, 48 were found to be harbouring a falciparum infection by conventional microscopy, 60 by conventional PCR and 60 by multiplex competitive real-time PCR. Nine further patients had a non-falciparum infection, and concordant species identifications were obtained by both conventional PCR and single real-time PCR. The major value of PCR-based methods, when compared to microscopical techniques, was to ascertain the negativity of a suspect sample. Moreover, real-time PCR allows simplification of the operating procedure, with a diagnosis being made within 2 h.

Topics: Animals; Benzothiazoles; Diamines; DNA, Protozoan; Humans; Malaria, Falciparum; Organic Chemicals; Parasitemia; Plasmodium falciparum; Polymerase Chain Reaction; Quinolines; Sensitivity and Specificity