sybr-green-i and Malaria--Falciparum

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

Owing to its fast and reliable assessment of parasite growth, the SYBR

Topics: Antimalarials; Benzothiazoles; Cell Survival; Diamines; DNA; Drug Evaluation, Preclinical; Erythrocytes; Fluorescent Dyes; Growth Inhibitors; Humans; Inhibitory Concentration 50; Malaria, Falciparum; Organic Chemicals; Parasitic Sensitivity Tests; Plasmodium falciparum; Quinolines; RNA; Spectrometry, Fluorescence

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

We have developed a convenient, robust and low-cost RNA detection system suitable for high-throughput applications. This system uses a highly specific sandwich hybridization to capture target RNA directly onto solid support, followed by on-site signal amplification via 2-dimensional, branched hybridizing chain polymerization through toehold-mediated strand displacement reaction. The assay uses SYBR Green to detect targets at concentrations as low as 1 pM, without involving nucleic acid purification or any enzymatic reaction, using ordinary oligonucleotides without modification or labeling. The system was demonstrated in the detection of malaria RNA in blood and GAPDH gene expression in cell lysate.

Topics: Benzothiazoles; Cell Line; Diamines; Fluorescent Dyes; Humans; Malaria, Falciparum; Nucleic Acid Hybridization; Organic Chemicals; Plasmodium falciparum; Quinolines; RNA; RNA, Protozoan

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

To investigate the antimalarial activity of four choline derivatives against Plasmodium falciparum 3D7 strain growth in vitro.. Four choline derivatives MD [N-dodecyl-N-(2-hydroxyethyl)-N,N- dimethyl ammonium bromide], ED [N-dodecyl-N-(2-hydroxyethyl)-N,N-diethyl ammonium bromide], MT [N-tetradecyl-N- (2-hydroxyethyl)-N,N-dimethyl ammonium bromide], and ET [N-tetradecyl-N-(2-hydroxyethyl)-N,N-diethyl ammonium bromide] were dissolved separately in DMSO at serial concentrations (1-10(5) µmol/L). The solutions were diluted by 1,000-fold with RPMI 1640 medium. 20 µl drug-containing medium and 80 µl P. falciparum-infected erythrocyte suspension (2% final hematocrit and 0.3%-0.5% parasitemia) were added to each well of microtiter plates. Drug effect on the in vitro growth of P. falciparum was measured by SYBR Green I method. The half maximal inhibitory concentration (IC50) was calculated from dose-response curves. Artemisinine served as positive control.. Artemisinine, MD, ED, MT, and ET showed different degrees of dose-dependent inhibition on P. falciparum growth. When the MD concentration was above 10 nmol/L, the inhibition rate increased significantly. Both ED and ET showed significant inhibitory effects at high concentrations, with inhibition rate of > 95% when their doses were > 10(4) nmol/L. The IC50 values of MD, ED, MT, and ET were 1 620, 33.9, 116, and 68.9 nmol/L, respectively, all significantly higher than that of artemisinine (5.7 nmol/L) (P < 0.05).. The four choline derivatives show certain antimalarial activity, which is lower than that of artemisinine. Among the four derivatives, ED has the strongest antimalarial activity against P. falciparum 3D7 strain.

Topics: Antimalarials; Artemisinins; Benzothiazoles; Choline; Diamines; Humans; Malaria, Falciparum; Organic Chemicals; Plasmodium falciparum; Quinolines

Malaria is caused by five Plasmodium species and transmitted by anopheline mosquitoes. It occurs in single and mixed infections. Mixed infection easily leads to misdiagnosis. Accurate detection of malaria species is vital. Therefore, the study was conducted to determine the level of mixed infection and misdiagnosis of malaria species in the study area using SYBR Green I-based real time PCR.. The study was conducted in seven health centres from North Gondar, north-west Ethiopia. The data of all febrile patients, who attended the outpatient department for malaria diagnosis, from October to December 2013, was recorded. Dried blood spots were prepared from 168 positive samples for molecular re-evaluation. Parasite DNA was extracted using a commercial kit and Plasmodium species were re-evaluated with SYBR Green I-based real time PCR to detect mixed infections and misdiagnosed mono-infections.. Among 7343 patients who were diagnosed for malaria in six study sites within the second quarter of the Ethiopian fiscal year (2013) 1802 (24.54%) were positive for malaria parasite. Out of this, 1,216 (67.48%) Plasmodium falciparum, 553 (30.68%) Plasmodium vivax and 33 (1.8%) mixed infections of both species were recorded. The result showed high prevalence of P. falciparum and P. vivax, but very low prevalence of mixed infections. Among 168 samples collected on dried blood spot 7 (4.17%) were P. vivax, 158 (94.05%) were P. falciparum and 3 (1.80%) were mixed infections of both species. After re-evaluation 10 (5.95%) P. vivax, 112 (66.67%) P. falciparum, 21 (12.50%) P. falciparum + P. vivax mixed infection, and 17 (10.12%) Plasmodium ovale positive rate was recorded. The re-evaluation showed high level of mixed infection, and misdiagnosis of P. ovale and P. vivax.. The result shows that P. falciparum prevalence is higher than P. vivax in the study area. The results, obtained from SYBR Green I-based real time PCR, indicated that the diagnosis efficiency of microscopy is very low for species-specific and mixed infection detection. Therefore, real time PCR-based species diagnosis should be applied for clinical diagnosis and quality control purposes in order to prevent the advent of drug resistant strains due to misdiagnosis and mistreatment.

Topics: Adolescent; Adult; Benzothiazoles; Child; Child, Preschool; Coinfection; Cross-Sectional Studies; Diamines; DNA, Protozoan; Dried Blood Spot Testing; Ethiopia; Female; Humans; Infant; Infant, Newborn; Malaria, Falciparum; Malaria, Vivax; Male; Organic Chemicals; Plasmodium falciparum; Plasmodium vivax; Prevalence; Quinolines; Real-Time Polymerase Chain Reaction; Young Adult

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

Performance of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR Green I fluorescence (MSF) drug sensitivity tests were directly compared using Plasmodium falciparum reference strains and fresh ex vivo isolates from Cambodia against a panel of standard anti-malarials. The objective was to determine which of these two common assays is more appropriate for studying drug susceptibility of "immediate ex vivo" (IEV) isolates, analysed without culture adaption, in a region of relatively low malaria transmission.. Using the HRP-2 and MSF methods, the 50% inhibitory concentration (IC50) values against a panel of malaria drugs were determined for P. falciparum reference clones (W2, D6, 3D7 and K1) and 41 IEV clinical isolates from an area of multidrug resistance in Cambodia. Comparison of the IC50 values from the two methods was made using Wilcoxon matched pair tests and Pearson's correlation. The lower limit of parasitaemia detection for both methods was determined for reference clones and IEV isolates. Since human white blood cell (WBC) DNA in clinical samples is known to reduce MSF assay sensitivity, SYBR Green I fluorescence linearity of P. falciparum samples spiked with WBCs was evaluated to assess the relative degree to which MSF sensitivity is reduced in clinical samples.. IC50 values correlated well between the HRP-2 and MSF methods when testing either P. falciparum reference clones or IEV isolates against 4-aminoquinolines (chloroquine, piperaquine and quinine) and the quinoline methanol mefloquine (Pearson r = 0.85-0.99 for reference clones and 0.56-0.84 for IEV isolates), whereas a weaker IC50 value correlation between methods was noted when testing artemisinins against reference clones and lack of correlation when testing IEV isolates. The HRP-2 ELISA produced a higher overall success rate (90% for producing IC50 best-fit sigmoidal curves), relative to only a 40% success rate for the MSF assay, when evaluating ex vivo Cambodian isolates. Reduced sensitivity of the MSF assay is likely due to an interference of WBCs in clinical samples.. For clinical samples not depleted of WBCs, HRP-2 ELISA is superior to the MSF assay at evaluating fresh P. falciparum field isolates with low parasitaemia (<0.2%) generally observed in Southeast Asia.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Protozoan; Antimalarials; Benzothiazoles; Cambodia; Diamines; Enzyme-Linked Immunosorbent Assay; Female; Fluorescence; Humans; Inhibitory Concentration 50; Malaria, Falciparum; Male; Middle Aged; Molecular Diagnostic Techniques; Organic Chemicals; Parasitic Sensitivity Tests; Plasmodium falciparum; Proteins; Protozoan Proteins; Quinolines; Staining and Labeling; Young Adult

In 2008, dihydroartemisinin (DHA)-piperaquine (PPQ) became the first-line treatment for uncomplicated Plasmodium falciparum malaria in western Cambodia. Recent reports of increased treatment failure rates after DHA-PPQ therapy in this region suggest that parasite resistance to DHA, PPQ, or both is now adversely affecting treatment. While artemisinin (ART) resistance is established in western Cambodia, there is no evidence of PPQ resistance. To monitor for resistance to PPQ and other antimalarials, we measured drug susceptibilities for parasites collected in 2011 and 2012 from Pursat, Preah Vihear, and Ratanakiri, in western, northern, and eastern Cambodia, respectively. Using a SYBR green I fluorescence assay, we calculated the ex vivo 50% inhibitory concentrations (IC50s) of 310 parasites to six antimalarials: chloroquine (CQ), mefloquine (MQ), quinine (QN), PPQ, artesunate (ATS), and DHA. Geometric mean IC50s (GMIC50s) for all drugs (except PPQ) were significantly higher in Pursat and Preah Vihear than in Ratanakiri (P ≤ 0.001). An increased copy number of P. falciparum mdr1 (pfmdr1), an MQ resistance marker, was more prevalent in Pursat and Preah Vihear than in Ratanakiri and was associated with higher GMIC50s for MQ, QN, ATS, and DHA. An increased copy number of a chromosome 5 region (X5r), a candidate PPQ resistance marker, was detected in Pursat but was not associated with reduced susceptibility to PPQ. The ex vivo IC50 and pfmdr1 copy number are important tools in the surveillance of multidrug-resistant (MDR) parasites in Cambodia. While MDR P. falciparum is prevalent in western and northern Cambodia, there is no evidence for PPQ resistance, suggesting that DHA-PPQ treatment failures result mainly from ART resistance.

Topics: Antimalarials; Artemisinins; Benzothiazoles; Biomarkers; Cambodia; Diamines; DNA Copy Number Variations; Drug Combinations; Drug Resistance, Multiple; Epidemiological Monitoring; Gene Expression; Humans; Malaria, Falciparum; Microscopy, Fluorescence; Multidrug Resistance-Associated Proteins; Organic Chemicals; Parasitic Sensitivity Tests; Plasmodium falciparum; Quinolines

Based on report of declining efficacy of chloroquine, Ghana shifted to the use of artemisinin-based combination therapy (ACT) in 2005 as the first-line anti-malarial drug. Since then, there has not been any major evaluation of the efficacy of anti-malarial drugs in Ghana in vitro. The sensitivity of Ghanaian Plasmodium falciparum isolates to anti-malarial drugs was, therefore, assessed and the data compared with that obtained prior to the change in the malaria treatment policy.. A SYBR Green 1 fluorescent-based in vitro drug sensitivity assay was used to assess the susceptibility of clinical isolates of P. falciparum to a panel of 12 anti-malarial drugs in three distinct eco-epidemiological zones in Ghana. The isolates were obtained from children visiting health facilities in sentinel sites located in Hohoe, Navrongo and Cape Coast municipalities. The concentration of anti-malarial drug inhibiting parasite growth by 50% (IC50) for each drug was estimated using the online program, ICEstimator.. Pooled results from all the sentinel sites indicated geometric mean IC50 values of 1.60, 3.80, 4.00, 4.56, 5.20, 6.11, 10.12, 28.32, 31.56, 93.60, 107.20, and 8952.50 nM for atovaquone, artesunate, dihydroartemisin, artemether, lumefantrine, amodiaquine, mefloquine, piperaquine, chloroquine, tafenoquine, quinine, and doxycycline, respectively. With reference to the literature threshold value indicative of resistance, the parasites showed resistance to all the test drugs except the artemisinin derivatives, atovaquone and to a lesser extent, lumefantrine. There was nearly a two-fold decrease in the IC50 value determined for chloroquine in this study compared to that determined in 2004 (57.56 nM). This observation is important, since it suggests a significant improvement in the efficacy of chloroquine, probably as a direct consequence of reduced drug pressure after cessation of its use. Compared to that measured prior to the change in treatment policy, significant elevation of artesunate IC50 value was observed. The results also suggest the existence of possible cross-resistance among some of the test drugs.. Ghanaian P. falciparum isolates, to some extent, have become susceptible to chloroquine in vitro, however the increasing trend in artesunate IC50 value observed should be of concern. Continuous monitoring of ACT in Ghana is recommended.

Topics: Antimalarials; Benzothiazoles; Child; Child, Preschool; Chloroquine; Diamines; Drug Resistance; Ghana; Humans; Infant; Inhibitory Concentration 50; Malaria, Falciparum; Organic Chemicals; Plasmodium falciparum; Quinolines

Emergence of artemisinin resistance in Cambodia highlights the importance of characterizing resistance to this class of drugs. Previously, intermediate levels of resistance in Plasmodium falciparum were generated in vitro for artelinic acid (AL) and artemisinin (QHS). Here we expanded on earlier selection efforts to produce levels of clinically relevant concentrations, and the resulting lines were characterized genotypically and phenotypically. Recrudescence assays determined the ability of resistant and parent lines to recover following exposure to clinically relevant levels of drugs. Interestingly, the parent clone (D6) tolerated up to 1,500 ng/ml QHS, but the resistant parasite, D6.QHS340×3, recovered following exposure to 2,400 ng/ml QHS. Resistant D6, W2, and TM91c235 parasites all exhibited elevated 50% inhibitory concentrations (IC(50)s) to multiple artemisinin drugs, with >3-fold resistance to QHS and AL; however, the degree of resistance obtained with standard methods was remarkably less than expected for parasite lines that recovered from 2,400-ng/ml drug pressure. A novel assay format with radiolabeled hypoxanthine demonstrated a greater degree of resistance in vitro than the standard SYBR green method. Analysis of merozoite number in resistant parasites found D6 and TM91c235 resistant progeny had significantly fewer merozoites than parent strains, whereas W2 resistant progeny had significantly more. Amplification of pfmdr1 increased proportionately to the increased drug levels tolerated by W2 and TM91c235, but not in resistant D6. In summary, we define the artemisinin resistance phenotype as a decrease in susceptibility to artemisinins along with the ability to recover from drug-induced dormancy following supraclinical concentrations of the drug.

Topics: Antimalarials; Artemisinins; Benzothiazoles; Cell Culture Techniques; Diamines; Dose-Response Relationship, Drug; Drug Resistance; Erythrocytes; Gene Dosage; Genotype; Humans; Hypoxanthine; Inhibitory Concentration 50; Malaria, Falciparum; Merozoites; Microscopy; Multidrug Resistance-Associated Proteins; Organic Chemicals; Parasitic Sensitivity Tests; Phenotype; Plasmodium falciparum; Quinolines; Recurrence

The pfmdr1 gene, which encodes P-glycoprotein homolog 1, has been shown to be a reliable marker of resistance for Plasmodium falciparum related to artesunate and mefloquine combination therapy. The aims of this study are to investigate the copy number of pfmdr1 in P. falciparum isolates collected from the 4 malaria-endemic areas of Thailand (Kanchanaburi, Mae Hongson, Ranong, and Tak) along the Thailand-Myanmar (Burma) border (Thai-Myanmar border) by using SYBR Green I and the standard method TaqMan real-time polymerase chain reaction (RT-PCR) and to compare the efficiency (sensitivity and specificity) of SYBR Green I with TaqMan RT-quantitative (q)PCR methods in determining pfmdr1 gene copy number. Ninety-six blood samples were collected onto filter paper from patients with uncomplicated falciparum malaria who attended malaria clinics in the Kanchanaburi (n  =  45), Mae Hongson (n  =  18), Ranong (n  =  11), and Tak (n  =  22) provinces in Thailand. Parasite genomic DNA was extracted from dried blood spots by using QIAcube™ automated sample preparation. Pfmdr1 gene copy number was determined by TaqMan (63 samples) and SYBR Green I (96 samples) real-time PCR. Seventy-one (74.0%), 14 (14.6%), 10 (10.4%), and 1 (1%) isolates carried 1, 2, 3, and 4 pfmdr1 gene copies, respectively. Forty-three of 48 (89.6%), 6 of 11 (54.5%), and 3 of 4 (75.0%) samples, respectively, showed agreement with results of 1, 2, and 3 pfmdr1 gene copies as determined by both methods. The efficiency of SYBR Green I in identifying pfmdr1 gene copy number was found to be significantly correlated with that of TaqMan. Considering its simplicity and relatively low cost, SYBR Green I RT-qPCR is therefore a promising alternative technique for the determination of pfmdr1 copy number.

Topics: Benzothiazoles; Diamines; DNA, Protozoan; Fluorescent Dyes; Gene Dosage; Humans; Malaria, Falciparum; Multidrug Resistance-Associated Proteins; Organic Chemicals; Plasmodium falciparum; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

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

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

Recently developed Sybr Green-based in vitro Plasmodium falciparum drug sensitivity assays provide an attractive alternative to current manual and automated methods. The present study evaluated flow cytometry measurement of DNA staining with Sybr Green in comparison with the P. falciparum lactate dehydrogenase assay, the tritiated hypoxanthine incorporation assay, a previously described Sybr Green based plate reader assay and light microscopy.. All assays were set up in standardized format in 96-well plates. The 50% inhibitory concentrations (IC50) of chloroquine, mefloquine and dihydroartemisinin against the laboratory adapted P. falciparum strains 3D7, E8B, W2mef and Dd2 were determined using each method.. The resolution achieved by flow cytometry allowed quantification of the increase in individual cell DNA content after an incubation period of only 24 h. Regression, and Bland and Altman analyses showed that the IC50 values determined using the flow cytometry assay after 24 h agreed well with those obtained using the hypoxanthine incorporation assay, the P. falciparum lactate dehydrogenase assay, the Sybr Green plate reader assay and light microscopy. However the values obtained with the flow cytometry assay after 48 h of incubation differed significantly from those obtained with the hypoxanthine incorporation assay, and the P. falciparum lactate dehydrogenase assay at low IC50 values, but agreed well with the Sybr Green plate reader assay and light microscopy.. Although flow cytometric equipment is expensive, the necessary reagents are inexpensive, the procedure is simple and rapid, and the cell volume required is minimal. This should allow field studies using fingerprick sample volumes.

Topics: Antimalarials; Benzothiazoles; Diamines; DNA, Protozoan; Dose-Response Relationship, Drug; Drug Resistance; Female; Flow Cytometry; Fluorescent Dyes; Humans; Inhibitory Concentration 50; L-Lactate Dehydrogenase; Malaria, Falciparum; Male; Microscopy; Organic Chemicals; Parasitic Sensitivity Tests; Plasmodium falciparum; Quinolines

Giemsa staining of thick blood smears remains the "gold standard" for detecting malaria. However, this method is not very good for diagnosing low-level infections. A method for the simultaneous staining of Plasmodium-parasitized culture and blood smears for both bright field and fluorescence was developed and its ability to improve detection efficiency tested.. A total of 22 nucleic acid-specific fluorescent dyes were tested for their ability to provide easily observable staining of Plasmodium falciparum-parasitized red blood cells following Giemsa staining.. Of the 14 dyes that demonstrated intense fluorescence staining, only SYBR Green 1, YOYO-1 and ethidum homodimer-2 could be detected using fluorescent microscopy, when cells were first stained with Giemsa. Giemsa staining was not effective when applied after the fluorescent dyes. SYBR Green 1 provided the best staining in the presence of Giemsa, as a very high percentage of the parasitized cells were simultaneously stained. When blood films were screened using fluorescence microscopy the parasites were more readily detectable due to the sharp contrast between the dark background and the specific, bright fluorescence produced by the parasites.. The dual staining method reported here allows fluorescence staining, which enhances the reader's ability to detect parasites under low parasitaemia conditions, coupled with the ability to examine the same cell under bright field conditions to detect the characteristic morphology of Plasmodium species that is observed with Giemsa staining.

Topics: Animals; Azure Stains; Benzothiazoles; Diamines; Erythrocytes; Fluorescent Dyes; Humans; Malaria, Falciparum; Microscopy, Fluorescence; Organic Chemicals; Plasmodium falciparum; Quinolines; Reproducibility of Results; 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

Plasmodium falciparum drug resistance is a major problem in malaria endemic areas. Molecular markers and in vitro tests have been developed to study and monitor drug resistance. However, none, used alone, can provide sufficient data concerning the level of drug resistance and to issue precise guidelines for drug use policies in endemic areas. We propose real-time PCR for the simultaneous detection of pfcrt and pfmdr1 genes mutations and to determine the half-maximal inhibitory response (IC(50)) of antimalarial drug. Using hybridization probes and SybrGreen technology on LightCycler instrument, point mutations of pfcrt and pfmdr1 genes have been successfully detected in 161 human blood samples and determination of IC values was applied to chloroquine-sensitive and chloroquine-resistant strains. Moreover, mixed infections caused by P. falciparum clones with wild-type or mutant alleles could be efficiency separated. The aim of this study was not to provide definitive data concerning the rate of mutations in an endemic area, but to describe a powerful method allowing the quantification of DNA for IC(50) determination and the detection of major pfmdr1 and pfcrt mutations.

Topics: Animals; Antimalarials; ATP-Binding Cassette Transporters; Benzothiazoles; Chloroquine; Diamines; DNA, Protozoan; Drug Resistance; Fluorescence Resonance Energy Transfer; Genetic Variation; Humans; Inhibitory Concentration 50; Malaria, Falciparum; Organic Chemicals; Plasmodium falciparum; Point Mutation; Polymerase Chain Reaction; Polymorphism, Genetic; Protozoan Proteins; Quinolines