Abstract
The malaria SYBR green assay, which is used to profile in vitro drug susceptibility of Plasmodium 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. falciparum laboratory 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.
INTRODUCTION
Malaria remains one of the most important preventable infectious diseases, with an estimated 124 to 283 million episodes and 584,000 deaths worldwide in 2013, according to the World Health Organization (WHO) malaria report 2014 (1). The emergence and spread of Plasmodium falciparum parasites that are resistant to antimalarial drugs pose a significant challenge to disease management and threaten the progress made in malaria control and the elimination agenda. The lack of an effective malaria vaccine to date has led to a reliance on chemotherapy for the management of infections (2, 3). Drug resistance surveillance is therefore critical to guiding malaria control programs.
In vitro methods have been used to monitor malaria drug resistance (4, 5). The techniques used for in vitro growth inhibition analyses in P. falciparum include radioisotope uptake (6), serological assays (7), fluorescence-based assays (8), and, more recently, ring-stage survival assays for artemisinin resistance testing (9, 10). The requirement of specialized equipment and supplies for radioisotope and serological assays makes them unsustainable in resource-constrained laboratories. Most of the laboratories that require these capabilities are located in areas that are endemic for malaria, where most of the disease occurs and prompt solutions are needed. Furthermore, even if deployable techniques were innovated, an account of P. falciparum drug susceptibility testing across laboratories underscores the variability in practice and reagents, impeding data comparisons across laboratories. In order to combat the issue of standardization, the Worldwide Antimalarial Resistance Network (WWARN) provides harmonized guidelines for antimalarial drug testing, including procedures, such as “Estimation of Plasmodium falciparum drug susceptibility by the SYBR green assay” (http://www.wwarn.org/tools-resources/procedures/estimation-plasmodium-falciparum-drug-susceptibility-sybrr-green-assay).
The original malaria SYBR green I-based fluorescence (SYBR green) assay has been described as simple, cost-effective, and reliable (8, 11). The technique has been utilized for in vitro and ex vivo profiling of field isolate susceptibility to antimalarials (12–14). However, there are conflicting published data on its performance in isolates with low parasitemia and high hematocrit levels (15–17). For example, both Chaorattanakawee et al. (15) and Vossen et al. (16) have demonstrated that the SYBR green assay may falter in its ability to accurately determine antimalarial drug sensitivity for samples from P. falciparum laboratory strains and field isolates possessing low parasitemia levels. As indicated by the data from Moneriz et al. (17), this may be a result of fluorometric interference from the hemoglobin with the SYBR green I dye during the assay readout. In assaying field isolates, these factors are critical, given that parasite density and anemia vary between samples collected from different patients. The parasite density and anemia statuses of individuals change as malaria disease progresses. These parameters are determined by several factors, including the host immunity, parasite virulence, and time point at which the patient seeks treatment (18, 19). Assessments of the SYBR green assay performance at various parasitemia and hematocrit levels are therefore warranted for guiding the application of the technique for use with field isolates, especially for an immediate ex vivo assay.
Extensive validation of the performance of the SYBR green assay technique has been done in laboratory clones whose parasitemia levels can be adjusted by the researcher (8, 20). In our initial validation of the SYBR green assay in laboratory clones, after testing an extensive drug panel, including antibiotics and antifolates, and comparing these results to the [3H]hypoxanthine assay, we obtained drug sensitivity profiles that were similar to those obtained by Smilkstein et al. (8). In a follow-up study, we described the effect of assay components, including drugs and white blood cells, on the assay background signal, with the purpose of clarifying the applicability of the technique for ex vivo drug testing (12). Since then, the SYBR green assay has been used successfully to determine the 50% inhibitory concentrations (IC50s) of clinical isolates in comparison with the histidine-rich protein-2 (HRP2) enzyme-linked immunosorbent assay (ELISA) and the [3H]hypoxanthine assay. Plouffe et al. (21) significantly increased the sensitivity and lowered the detection limit of the SYBR green assay through augmentation of the lysis buffer components, SYBR green I dye concentration, and incubation time before plate reading. More specifically, Plouffe et al. (21) increased the lysis buffer components found in the original Smilkstein et al. (8) version to 5 mM EDTA (2-fold increase), 0.16% saponin (wt/vol) (20-fold increase), 1.6% Triton X-100 (vol/vol) (20-fold increase), and 10× SYBR green I dye concentration (10-fold increase). Additionally, incubation of the parasite culture in the presence of the lysis buffer was increased from 1 h to 24 h. Taken together, these changes synergistically allowed for increased human and red blood cell lysis, increased exposure of parasitic DNA, and increased binding of SYBR green I dye to the parasite DNA. The WWARN malaria SYBR green I-based fluorescence assay standardized procedure is similar to the method of Plouffe et al. (21); however, 0.016% (wt/vol) saponin (2-fold increase compared to Smilkstein et al. [8]) and 20× SYBR green I dye concentration (20-fold increase) are used (22). The WWARN procedure, adopted in 2011, has been used for 96- and 384-well malaria drug sensitivity screening of laboratory and field isolates by the Walter Reed Army Institute of Research since 2010 and at the U.S. Army Medical Research Unit, Kenya, since 2011, with great success. In spite of these modifications, conflicting data from other laboratories have suggested that the original method described by Smilkstein et al. (8) may be flawed. For example, suggestions that the presence of hemoglobin may affect fluorescence-based assays due to its wide absorption spectrum, which can interfere with the emission of SYBR green I dye, have been made (17). Also, Quashie et al. (23) proposed that detergents found in lysis solutions may have a negative effect on DNA detection by fluorophores. Assay sensitivity in isolates with low parasitemia levels has also been debated (16, 24).
At this time, all current versions of the SYBR green assay, including the WWARN standardized procedure, use a set initial parasitemia level as a requirement for assay standardization. For example, the version of the assay by Smilkstein et al. (8) uses 1% parasitemia for initiation of the 48-h drug sensitivity assays (8). Plouffe et al. (21) uses 0.3% parasitemia for the 72-h assay, while the WWARN version (22) uses 0.3% and 0.15% parasitemia for the 72- and 96-h assays, respectively. This is troublesome, as Chaorattanakawee et al. (15) has reported that about 49% of Cambodian and 22% of Kenyan natural infections had parasitemia levels of <0.2%, depicting large variations in parasite density across the different transmission regions (15). However, with the limit of detection of 0.20% in white blood cell (WBC)-free culture and 0.55% in whole blood (16), the original SYBR green assay technique is inapplicable to screening of most of the isolates found in these regions by immediate ex vivo technique (i.e., patient to assay plate without the need for in vitro culture before assaying). As the SYBR green I drug sensitivity assay based on the WWARN procedure continues to be widely adopted by field-based laboratories, we examined this assay for its sensitivity and ability to accurately determine IC50s in samples with a wide range of initial parasitemia levels. This improvement in accuracy is of particular importance, as the technique continues to be used in immediate ex vivo assaying of field isolates obtained from individuals with naturally acquired malaria infections, whose parasitemia levels are highly varied and possibly too low to be included in other currently used versions of the SYBR green assay for drug sensitivity determination.
MATERIALS AND METHODS
This study was approved by the Kenya Medical Research Institute (KEMRI) and Walter Reed Army Institute of Research (WRAIR) institutional review boards (protocol numbers KEMRI 1330 and WRAIR 1384, respectively). Subject inclusion/exclusion criteria, sample collection, and transportation were as previously published (12). The red blood cells and serum used in P. falciparum culture were obtained from nonimmune individuals under protocol WRAIR 1919.
Drug.
Chloroquine diphosphate, mefloquine, artemisinin, artesunate, and atovaquone were obtained from the WRAIR, Silver Spring, MD.
P. falciparum culture.
P. falciparum field isolates were obtained from ongoing epidemiology studies of malaria drug resistance patterns in Kenya, collected under protocol numbers KEMRI 1330 and WRAIR 1384. Reference P. falciparum clones D6 and C235 were cultured at the WRAIR (Silver Spring, MD). Malaria field isolate and routine reference clone culture was performed as previously described (20, 25).
Assessment of SYBR green I fluorescence linearity.
Fluorescence linearity of the SYBR green assay was verified over a range of 0 to 5% parasitemia (at 2% hematocrit), as determined by Giemsa staining of D6 parasites, as previously described (20, 25). The assay was performed at the WRAIR in 384-well optical-bottom Nunc plates using the Tecan Freedom EVO 150 with MCA-96 and RoMa automated liquid handling workstation (Tecan US, Inc., Durham, NC, USA).
Drug dilution and preparation of predosed drug plates.
Drug dilutions of between 3,876 and 1.89 nM were made on 96-well culture plates (catalog no. 167008; Nunc, Inc., Roskilde, Denmark) by loading the working concentration of 3,876 nM onto the first column of wells (1A to 1H), followed by 2-fold serial dilutions across 12 wells using the Biomek FXP automated laboratory workstation (Beckman Coulter, Inc., Fullerton, CA, USA). For both culture-adapted and immediate ex vivo assays, 3.12 μl of drug aliquots was made from 96-well microculture plates and transferred to 384-well plates (catalog no. 142761; Nunc, Inc.). For experiments performed at the WRAIR, drug dilutions on 96-well plates and transfers to 384-well optical-bottom Nunc plates were performed using the Tecan Freedom EVO 150 with MCA-96 and RoMa automated liquid handling workstation.
WWARN SYBR green assay using range of initial parasitemia levels.
Experiments to assess the sensitivities of D6 and C235 laboratory strains to chloroquine (CQ), mefloquine (MQ), artesunate (AR), and atovaquone (ATOV) were performed at the Division of Experimental Therapeutics, WRAIR, using the Tecan Freedom EVO 150 with MCA-96 and RoMa automated liquid handling workstation. Experiments analyzing immediate ex vivo susceptibility of field isolates to chloroquine were performed at the Malaria Drug Resistance Laboratory of the Kenya Medical Research Institute (KEMRI)/U.S. Army Medical Research Unit, Kenya, using the Biomek FXP automated laboratory workstation. The in vitro malaria drug sensitivity SYBR green assay was performed, according to the WWARN procedure (22), with the exception of being scaled to 384-well microtiter plates, as in Plouffe et al. (21). The assay was initiated by the transfer of parasite sample (25 μl) onto the 384-well microculture predosed drug plates by a semiautomated workstation. The field isolates were tested ex vivo (no prior laboratory adaptation). The parasitemia level (as a percentage) was determined as the number of infected cells per total number of red blood cells. The field samples with >1% parasitemia and culture-adapted D6 and C235 clones with 3 to 8% parasitemia were diluted to 0.6%, 0.3%, 0.15%, 0.075%, 0.038%, 0.019%, 0.0094%, 0.0044%, and 0.0023% parasitemia prior to drug testing. The plates were then incubated for 72 h, after which 25 μl of lysis buffer containing SYBR green I dye was added to the plates. The plates were then stored in the dark at ambient temperature for 24 h. Next, the fluorescence was determined using a plate reader at an excitation wavelength of 485 nm and emission wavelength of 535 nm, as previously described (20). The inhibition of parasite replication was quantified and the IC50 for each drug calculated as in reference 20, generating a sigmoidal concentration-response curve (variable slope), with log-transformed drug concentrations on the x axis and relative fluorescence units (RFUs) on the y axis (GraphPad Prism for Windows, version 5.0; GraphPad Software, Inc., San Diego, CA) (11, 20). The effects of various parasitemia levels on drug response were assessed by one-way analysis of variance.
Assessment of field performance of the WWARN standardized SYBR green assay.
P. falciparum isolates obtained between 2013 and 2014 were placed into the WWARN standardized SYBR green assay within 6 h postphlebotomy, without culture adaptation (immediate ex vivo). Blood samples with >0.3% parasitemia were adjusted to 0.3% parasitemia at 2% hematocrit, per the WWARN standardized procedure. Those samples with ≤0.3% parasitemia were tested unadjusted at 2% hematocrit. The drugs examined included chloroquine, mefloquine, and artesunate. The IC50s generated were then evaluated and compared between samples with initial parasitemia levels of <0.3% and 0.3%.
Effect of various hematocrit levels on WWARN standardized SYBR green assay.
To assess the effect of various hematocrit levels on the WWARN standardized procedure for in vitro malaria drug sensitivity test using the SYBR green assay, the chloroquine susceptibility of D6 was tested at 0.3% parasitemia while changing hematocrit levels between 0.5%, 1%, and 4%, parallel to the conventional 2% hematocrit level, and the IC50s were determined as described above.
Determination of assay quality (Z′).
The statistical test published by Zhang et al. (26) was used to determine assay quality. Based on this statistical test, the separation band (S), represented by the difference between the mean of the signal values minus three times the standard deviation and the mean of the background values plus three times the standard deviation, was calculated for each parasitemia level. Further, the corresponding difference between the absolute signal means and absolute background means, called the dynamic range (R), was calculated. The Z′ was then calculated as the S/R ratio, providing values between 0.5 and 1.0 (excellent assay), 0 and 0.5 (marginal assay), or <0 (poor assay).
RESULTS
Sensitivity of the WWARN SYBR green assay standardized procedure.
The fluorescence linearity of the SYBR green I assay was verified over a range of 0 to 5% parasitemia (at 2% hematocrit), as determined by Giemsa staining of D6 parasites. One-way analysis of variance (ANOVA) with multiple-comparison Tukey's test analysis was performed on the data obtained. A linear relationship between SYBR green I fluorescence and parasitemia level was observed (Fig. 1). Next, the sensitivity of the assay was examined by determining the detection limit (DL). The DL was defined as the lowest parasitemia level in a well that was detected above noninfected erythrocyte staining. The WWARN SYBR green assay performed in a 384-well format on the Tecan robot yielded a DL of 0.019% (Fig. 1).
FIG 1.
Assessment of fluorescence linearity. The data are presented as the means ± the standard deviations of the results from at least duplicate samples. The data were analyzed by linear regression.
To further assess the sensitivity and performance of the WWARN standardized procedure for in vitro malaria drug sensitivity assay using the SYBR green I assay for the detection of parasite replication at various antimalarial doses and parasitemia levels, relative fluorescence unit (RFU) readouts for the chloroquine-sensitive D6 strain were obtained across the sigmoidal dose-response curve for chloroquine. Chloroquine was tested at concentrations ranging from 3,876 to 1.89 nM, with parasitemia levels ranging from 1 to 0.0023% over a 72-h incubation period. A level of 1% parasitemia was tested, due to its usage as the starting parasitemia level recommended by Smilkstein et al. (8) for the original malaria SYBR green I-based fluorescence assay and the traditional [3H]hypoxanthine assay (20). Serial dilutions starting from 0.6% parasitemia to 0.0023% parasitemia were made, as the highest parasitemia level represents a 2-fold increase in the recommended parasitemia level for the WWARN SYBR green I procedure for the 72-h assays. Figure 2 shows the RFU counts for the D6 strain in response to chloroquine at different parasitemia levels. As expected, parasites cultured in the absence of chloroquine at the highest parasitemia level had the highest RFU count, decreasing with the reduction in parasitemia level and the drug dose. As the parasitemia level incrementally decreased by half starting from 0.6% to 0.019%, there was a proportionate 50% decrease in the RFU. The highest initial parasitemia level tested (1%) did not proportionally increase its upper asymptote of the curve, unlike the other initial parasitemia levels tested.
FIG 2.
Chloroquine dose-response comparison using WWARN SYBR green I procedure. Chloroquine dose-response curves at different starting parasitemia were generated. The error bars represent standard deviations for the results from 10 replicate assays at each parasitemia level tested.
Assay success rate with different initial parasitemia levels.
In order to access the assess the success rate of the WWARN standardized procedure using different initial parasitemia levels, malaria laboratory strains and field isolates were subjected to antimalarial compounds, and sigmoidal dose-response curves were generated. The parasitemia levels for both the D6 and C235 strains and field isolate parasites were adjusted to 1% and 0.6% and then serially diluted to various concentrations. At each dilution, three to 11 replicates of the assay were performed for both the D6 and C235 strains in the presence of chloroquine, mefloquine, artemisinin, and atovaquone. The field isolates, for which one to four replicates were done due to limitations in the amount of sample available for each, were tested against chloroquine. The success rate of the assay was determined by the proportion of successful replicate assays at each dilution. Successful assays produced data sets across various doses that were used to generate sigmoidal dose-response curves in order to compute the IC50 at each dilution. Tables 1 and 2 and Table S1 in the supplemental material show the parasitemia levels for each assay, the number of successful replicate assays, and IC50s (mean ± standard deviation) for D6, C235, and the ex vivo field isolates, respectively. At higher parasitemia levels, most of the replicates were successful and were used to compute the IC50s. For example, D6 treated with chloroquine at 0.6% and 0.3% parasitemia had 8/11 and 9/11 successful replicates, respectively, and parasitemia dilutions of 0.15 to 0.038% produced 10/11 successful replicates. As the level of parasitemia was reduced, the number of successful replicates decreased, with only 4 successful replicates at 0.0023% parasitemia. Similar trends were observed for all the drugs evaluated against D6 and C235. For the immediate ex vivo field isolates, successful assay replicates of >75% were seen between 0.038 and 1% initial parasitemia and reduced to 0 to 50% for initial parasitemia levels of ≤0.019%.
TABLE 1.
Comparison of antimalarial IC50s for D6 determined using different starting parasitemia levels
| Drug (no. of replicates) | Data by % parasitemiaa |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1.00 |
0.60 |
0.30 |
0.15 |
0.075 |
0.038 |
0.019 |
0.0094 |
0.0044 |
0.0023 |
|||||||||||
| n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | |
| Chloroquine (11) | 11 | 22.4 ± 6.4 | 8 | 18.3 ± 7.4 | 9 | 13.8 ± 4.3 | 10 | 9 ± 4.3 | 10 | 10 ± 2.8 | 10 | 9.2 ± 1.6 | 6 | 9.8 ± 2.1 | 5 | 9.1 ± 2.5 | 5 | 8.4 ± 3.3 | 4 | 10.8 ± 4.1 |
| Mefloquine (6) | 4 | 16.8 ± 5.8 | 6 | 14.3 ± 4.6 | 6 | 12.1 ± 4.6 | 5 | 11.3 ± 2.9 | 5 | 10.8 ± 2.2 | 4 | 10.4 ± 3.9 | 3 | 15.2 ± 5.8 | 2 | 13.3 ± 10.6 | 2 | 16.9 ± 15 | ND | |
| Artemisinin (3) | 3 | 11.9 ± 1.4 | 3 | 10.7 ± 1.4 | 3 | 9.9 ± 6.0 | 3 | 6.1 ± 3.2 | 3 | 5.3 ± 2.8 | 3 | 5.4 ± 3.2 | 3 | 4.6 ± 2.5 | ND | 2 | 5.6 ± 0.7 | 2 | 0.5 ± 0.7 | |
| Atovaquone (3) | 3 | 0.4 ± 0.3 | 3 | 0.2 ± 0.3 | 2 | 0.2 ± 0.01 | 3 | 0.4 ± 0.3 | 3 | 0.3 ± 0.3 | 2 | 0.3 ± 0.3 | 3 | 0.1 ± 0.1 | 1 | 0.3 | ND | ND | ||
All IC50 data are expressed as the mean ± SD in nanomoles. n, number of successful replicates; ND, data not converged and IC50 could not be determined.
TABLE 2.
Comparison of chloroquine IC50s for D6 and field isolates determined using different starting parasitemia levels
| Isolate (no. of replicates) | Data by % parasitemiaa |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 |
0.6 |
0.3 |
0.15 |
0.075 |
0.038 |
0.019 |
0.0094 |
0.0044 |
0.0023 |
|||||||||||
| n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | n | IC50 | |
| D6 (4) | 4 | 22.4 ± 6 | 4 | 18.3 ± 7.4 | 4 | 13.8 ± 4.2 | 4 | 8.9 ± 4.3 | 4 | 9.8 ± 2.8 | 4 | 9.2 ± 1.6 | 4 | 9.8 ± 2.1 | 2 | 9.1 ± 2.5 | 3 | 8.4 ± 3.3 | 2 | 10.8 ± 4 |
| Field isolate 1 (4) | 4 | 104.2 ± 13.8 | 4 | 87.5 ± 9.5 | 4 | 67.6 ± 2.5 | 3 | 65.5 ± 9.9 | 4 | 67.8 ± 2.3 | 2 | 39.9 ± 2.1 | 2 | 42.6 ± 9.1 | 2 | 50.4 ± 10.3 | ND | ND | ||
| Field isolate 2 (4) | NT | 3 | 17.3 ± 4.3 | 4 | 8.0 ± 01 | 4 | 5.5 ± 1.5 | NT | 3 | 5.03 ± 2.8 | 1 | 3.9 | ND | ND | ND | |||||
| Field isolate 3 (4) | NT | 4 | 5.6 ± 4.8 | 1 | 7.872 | 3 | 9.3 ± 1.7 | NT | 3 | 3.4 ± 0.4 | ND | ND | ND | ND | ||||||
| Field isolate 4 (4) | NT | 3 | 10.9 ± 3.2 | 1 | 9.4 | 3 | 3.1 ± 9.2 | NT | 3 | 3.6 ± 0.3 | ND | ND | ND | ND | ||||||
| Field isolate 5 (1) | NT | 1 | 9.3 | 1 | 2.3 | 1 | 14.2 | NT | 1 | 14.4 | ND | ND | ND | ND | ||||||
| Field isolate 6 (1) | NT | 1 | 9.0 | 1 | 3.8 | 1 | 5.7 | NT | 1 | 6.4 | ND | ND | ND | ND | ||||||
All IC50 data are expressed as the mean ± SD in nanomoles. n, number of successful replicates; ND, data not converged and IC50 could not be determined; NT, not tested, since there was not enough clinical sample.
Assay variation in IC50 determinations at different starting parasitemia levels.
The IC50s produced at each dilution were analyzed in order to assess the ability to produce accurate IC50s across the range of parasitemia levels tested. One-way ANOVA with multiple-comparison Tukey's test analysis was performed on the data obtained. For chloroquine-treated D6, the IC50s decreased as the parasitemia level was reduced from 1% to 0.3%. From 0.15% to 0.0023% parasitemia, however, the IC50s remained relatively constant (Table 1). No statistically significant changes in IC50s occurred in a pairwise comparison with each other. C235 displayed results equivalent to those of the D6 strain. Overall, there were no statistically significant differences in the IC50s generated from any of the initial parasitemia levels tested within the range of 0.0375% to 0.6% for both laboratory stains tested, regardless of the drug used. For the field isolates, the IC50s also decreased as the parasitemia level was reduced from 1% to 0.3%. Interestingly, however, with the field isolates, the IC50s remained constant from 0.3% to ≤0.075% in some samples (Table 2). The final two dilutions did not produce any IC50s. It is worth noting that the IC50s at parasitemia levels of 0.6% and 1% were somewhat elevated compared to those with the other parasitemia levels for both the laboratory strains and the field isolates.
The effects of parasitemia variation on assay robustness.
To assess the robustness of the WWARN procedure for measuring drug effect, the Z′ was computed using a chloroquine-treated sample and nontreated field isolate samples with various parasitemia levels. Assays conducted with serially diluted P. falciparum parasitemia levels in a 72-h incubation in drug-free medium (which represents the signal value) had a median value of 55,244 RFUs (95% confidence interval [CI], 48,830 to 62,500 RFUs) for 1% parasitemia, decreasing to 3,343 RFUs (95% CI, 2,895 to 3,868 RFUs) for 0.009% parasitemia. The highest-drug (kill)-concentration wells, which were incubated for 72 h (which represents the background signal), showed a median value of 4,479 RFUs (95% CI, 3,786 to 5,299 RFUs) for 1% parasitemia, decreasing to 1,713 RFUs (95% CI, 1,905.5 to 3,240) for 0.009% parasitemia. Table 3 summarizes the WWARN standardized procedure robustness signal/noise ratio and Z′ at various immediate ex vivo parasitemia levels. Parasitemia levels of >0.075% demonstrated a Z′ between 0.629 and 0.871, categorized as excellent assays, while those with parasitemia levels of <0.0385% had Z′ of <0.5, which were categorized as marginal. The signal-to-noise ratios for assays with parasitemia levels of >0.075% were >5, which decreased to <5 for parasitemia levels of <0.0385%.
TABLE 3.
Determination of assay quality for the WWARN SYBR green I standardized procedure for field isolates with different starting parasitemia levels
| % initial parasitemia | Signal/noise ratio | Assay quality (Z′) |
|---|---|---|
| 0.009 | 1.95 | 0.296 |
| 0.018 | 1.82 | 0.128 |
| 0.038 | 2.88 | 0.392 |
| 0.075 | 5.66 | 0.871 |
| 0.15 | 10.32 | 0.758 |
| 0.3 | 14.01 | 0.629 |
| 0.6 | 15.21 | 0.786 |
| 1 | 12.33 | 0.761 |
The effects of hematocrit variation on assay quality.
In order to determine the effect of hematocrit variation on assay quality, the susceptibilities of D6 and selected field isolates to chloroquine were tested at various hematocrit levels in a standard 72-h assay. A parasitemia level of 0.3% was selected as the starting parasitemia level, since it is the standardized starting parasitemia level for the WWARN procedure. The hematocrit levels examined included 0.5%, 1%, 2%, and 4%, with 2% serving as the WWARN procedure standard. Across these four hematocrit levels, the highest RFUs, which correspond to the lowest concentration of chloroquine, were comparable (Fig. 3). This similarity among the various hematocrit concentrations was observed across the chloroquine dose range up to a concentration of 61 nM, at which no growth occurred. Importantly, the slopes of the sigmoidal curves, which are useful in the IC50 determination, were indistinct. Subsequently, the IC50s for both D6 and the field isolates at the various hematocrit levels, including 2%, which is the most used concentration, were statistically comparable, as determined by one-way ANOVA with multiple-comparison Tukey's test analysis (Table 4).
FIG 3.
Comparison of chloroquine dose-response curves at different starting hematocrit levels for D6. The error bars represent the standard deviations for the results from 10 replicate assays at each hematocrit level.
TABLE 4.
D6 and field isolate IC50s at various hematocrit levels
| % hematocrit | IC50 (mean ± SD) (nM) for: |
|
|---|---|---|
| D6 | Field isolate | |
| 4 | 18.78 ± 3.74 | 47.91 ± 12.79 |
| 2 (standard) | 18.80 ± 5.0 | 55.47 ± 2.13 |
| 1 | 15.14 ± 1.45 | 49.78 ± 8.72 |
| 0.5 | 14.81 ± 2.27 | 56.5 ± 1.16 |
Field testing results of the assay using different initial parasitemia levels.
Comparisons of the IC50s for chloroquine, mefloquine, and artesunate were examined for field isolates collected at >0.3% parasitemia and adjusted to 0.3% parasitemia before testing and for those arriving with <0.3% parasitemia (range, 0.017% to 0.3%) that were tested unadjusted. Statistical analyses were performed using an unpaired t test with Welch's correction for comparisons of the IC50s between the two groups and contingency table analyses by Fisher's exact test for comparisons of assay success. There was no significant difference in the IC50s for each of the drugs for the <0.3% and 0.3% parasitemia sample groups (P > 0.05, Fig. 4). Analysis of assay success among the two parasitemia ranges showed no significant difference in assay success rates among the two categories of parasitemia (P > 0.05).
FIG 4.
Comparison of IC50s for field isolates at standard 0.3% parasitemia versus lower parasitemia levels. The data are represented as the median IC50s and the interquartile range. t is the value derived from the unpaired t test.
DISCUSSION
This is the first report, to our knowledge, on the use of the WWARN standardized procedure for in vitro malaria drug sensitivity using the SYBR green I assay to profile drug responses in field isolates by 384-well immediate ex vivo testing. Enhancement of the WWARN assay involved increased concentration of detergents, dye, duration of culture, and labeling, based on modifications to the method of Plouffe et al. (21). These modifications enabled enhanced sensitivity that was needed to robustly conduct high-throughput screening of a diverse array of chemical entities in 384-well plate formats. In this study, the 384-well WWARN SYBR green assay procedure displays a 4-fold increase in sensitivity when the assay detection limit is compared to the original SYBR green assay performed in a 96-well format using the Biomek 2000 robot (20) (Fig. 1). The linearity of the assay was confirmed upon analysis of sigmoidal dose-response curves of chloroquine-treated D6 parasites in which the SYBR green assay was initiated with different starting parasitemia levels. More specifically, the increases in starting parasitemia levels were proportional to increases in relative fluorescence units (RFUs), with the exception of those in some of the extreme upper and lower parasitemia levels tested (Fig. 2). The inconsistency at 1% parasitemia may indicate an inoculum effect (27) or signal saturation (i.e., the absence of enough SYBR green dye to appropriately label all of the parasitic DNA present).
Changing the initial parasitemia levels for D6, C235, and the field isolates treated with chloroquine did not significantly alter the mean IC50s (Table 1, Table S1 in the supplemental material, and Table 2, respectively). More specifically, the IC50s displayed comparable results for laboratory strains in the range of 0.0375% to 0.6% parasitemia and for field isolates in the range of 0.075% to 0.6% parasitemia. The detection limit of 0.019% determined from the sensitivity analysis of the WWARN SYBR green assay (Fig. 1 and 2) and the 0.0375% to 0.6% range obtained from the comparison of chloroquine dose-response curves at different starting parasitemia levels in the WWARN assay reinforce one another, further validating the lower limit of the 0.0375% to 0.6% range indicated for D6 and C235. Previous studies have associated higher baseline parasitemia levels with elevation in IC50s (15). Similarly, the IC50s in the current study for D6, C235, and field isolates at parasitemia levels of 0.6 and 1% were somewhat elevated compared to those of the other parasitemia levels tested, as explained above. The number of successful assays remained generally stable between initial parasitemia levels ranging from 0.038% to 1%. For initial parasitemias below this level (0.002% to 0.018%), the percentage of successful assays dropped dramatically to ≤50%, indicating reduced reliability in the ≤0.018% parasitemic samples. Interestingly, for assays that worked with parasitemia levels of ≤0.018%, the IC50s were often comparable to those observed at parasitemia levels ranging from 0.038% to 0.15%, therefore underscoring the sensitivity and accuracy of the WWARN standardized procedure. Similar trends were observed with other drugs tested, including mefloquine, artemisinin, and atovaquone (Table 1; see also Table S1 in the supplemental material).
For most analytical assays, the Z′, described by Zhang et al. (26), is useful for quantifying the robustness of a high-throughput assay. The WWARN assay attained Z′ values between 0.629 and 0.871 for all field sample parasitemia levels of >0.075%. This indicates that the WWARN assay is robust and high quality within this range of parasitemia, which directly correlates to the consistent IC50s determined and the high success rate of the assay within the range of 0.075% to 0.6% initial field sample parasitemia. Further, a wider RFU range of >10-fold difference was attained in a comparison of signal from untreated wells and background from highest-drug (kill)-concentration wells, contrary to previously reported RFU signal ranges (12, 16). This allowed for easy fitting of dose-response curves and accurate depiction of IC50s across parasitemia levels. The higher initial parasitemia ranges consisting of 0.15% to 1% and initial parasitemia ranges comprising 0.002% to 0.019% had signal-to-noise ratio ranges of 10 to 15 and 2 to 5, respectively.
Furthermore, a range of hematocrit levels commonly used in antimalarial drug sensitivity testing showed uniformity in the production of sigmoidal dose-response curves for both D6 and field samples in the presence of chloroquine. Using the original SYBR green assay by Smilkstein et al. (8), a subsequent study by Moneriz et al. (17) showed a direct correlation between increases in hematocrit and decreases in RFU. A hematocrit level of 1 to 5% was associated with potentially significant decreases in fluorescence in the malaria SYBR green assay compared to those of control samples that contained equivalent malarial DNA in the absence of hemoglobin, suggesting a heavy interference of hemoglobin. With improved assay quality, our study showed that hematocrit levels commonly used in malaria parasite culture do not affect IC50 determination under the WWARN SYBR green assay conditions.
Most studies investigating the drug susceptibilities of field isolates utilize hospital-based enrollment arms as the source of specimens, in which clinical symptoms are listed as one of the key inclusion criteria. However, the parasitemia threshold for clinical symptoms or its correlation with severity of disease across transmission regions is still a matter of debate (18, 28). For Kenya, our ongoing epidemiology study of malaria and drug sensitivity patterns has revealed a median parasitemia level of 1.4% (interquartile range, 0.2 to 3.1%; n = 1,985), with diverse regional variation. About 75% of these isolates had a parasitemia level of ≥0.15%. Similar studies in Southeast Asia have shown that 51% of the natural infections are at ≥0.2% parasitemia (24). Findings from this study show that by ex vivo assaying, the success rates among isolates tested at parasitemia levels between 0.038 and 0.3% did not differ significantly from those of isolates at >0.3% parasitemia that were diluted to 0.3% prior to testing (Fig. 4 and Table 2). The wide range of starting parasitemia levels for the WWARN SYBR green assay is necessary for malaria field surveillance efforts in which isolates typically have various low levels of parasitemia. Thus, the assay is particularly suitable for the immediate ex vivo assaying of malaria field isolate drug sensitivity.
The WWARN standardized procedure for determining in vitro malaria drug sensitivity using the SYBR green I assay appears to be well suited for determining malaria dose-response curves of field isolates with various initial parasitemia levels, based on the assay's success rate, low variability, and its robustness (via Z′ analysis) within the range of 0.075% to 0.6% parasitemia. Changing the initial parasitemia levels within the detection range of the assay does not significantly alter the determined IC50s. The WWARN SYBR green assay enables IC50 determination in assays at various hematocrit levels commonly used in malaria parasite culture and drug testing. Augmentations to the original malaria SYBR green I-based fluorescence assay have significantly increased the technique's accuracy and applicability for assaying malaria field isolates with initial parasitemia levels too low for testing with the original method. The reproducibility of the assay across a wide range of initial parasitemia levels at low cost heralds the technique's field expedience as a surveillance tool.
Supplementary Material
ACKNOWLEDGMENTS
We thank the patients who enrolled in this study and all clinical staff serving at all our field sites. We thank our colleagues in MDR for their contributions and support for this work. We also thank M. A. J. Sheila Johnson for manuscript review and editing.
The opinions and assertions contained herein are the private opinions of the authors and do not necessarily reflect the official policy or positions of the U.S. Army Medical Research Directorate-Kenya, the U.S. Department of the Army, the U.S. Department of Defense, or the U.S. Government. This work was supported by the Armed Forces Health Surveillance Center, a division of Global Emerging Infections Surveillance and Response System (GEIS) operations.
We declare no conflicts of interest.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.00527-15.
REFERENCES
- 1.WHO. 2014. World malaria report 2014. World Health Organization, Geneva, Switzerland: http://www.who.int/malaria/publications/world_malaria_report_2014/wmr-2014-no-profiles.pdf?ua=1. [Google Scholar]
- 2.Bejon P, Lusingu J, Olotu A, Leach A, Lievens M, Vekemans J, Mshamu S, Lang T, Gould J, Dubois MC, Demoitie MA, Stallaert JF, Vansadia P, Carter T, Njuguna P, Awuondo KO, Malabeja A, Abdul O, Gesase S, Mturi N, Drakeley CJ, Savarese B, Villafana T, Ballou WR, Cohen J, Riley EM, Lemnge MM, Marsh K, von Seidlein L. 2008. Efficacy of RTS,S/AS01E vaccine against malaria in children 5 to 17 months of age. N Engl J Med 359:2521–2532. doi: 10.1056/NEJMoa0807381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Girard MP, Reed ZH, Friede M, Kieny MP. 2007. A review of human vaccine research and development: malaria. Vaccine 25:1567–1580. doi: 10.1016/j.vaccine.2006.09.074. [DOI] [PubMed] [Google Scholar]
- 4.Bacon DJ, Jambou R, Fandeur T, Le Bras J, Wongsrichanalai C, Fukuda MM, Ringwald P, Sibley CH, Kyle DE. 2007. World Antimalarial Resistance Network (WARN) II: in vitro antimalarial drug susceptibility. Malar J 6:120. doi: 10.1186/1475-2875-6-120. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Quashie NB, Duah NO, Abuaku B, Koram KA. 2007. The in-vitro susceptibilities of Ghanaian Plasmodium falciparum to antimalarial drugs. Ann Trop Med Parasitol 101:391–398. doi: 10.1179/136485907X176553. [DOI] [PubMed] [Google Scholar]
- 6.Desjardins RE, Canfield CJ, Haynes JD, Chulay JD. 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16:710–718. doi: 10.1128/AAC.16.6.710. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Noedl H, Bronnert J, Yingyuen K, Attlmayr B, Kollaritsch H, Fukuda M. 2005. Simple histidine-rich protein 2 double-site sandwich enzyme-linked immunosorbent assay for use in malaria drug sensitivity testing. Antimicrob Agents Chemother 49:3575–3577. doi: 10.1128/AAC.49.8.3575-3577.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M. 2004. Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrob Agents Chemother 48:1803–1806. doi: 10.1128/AAC.48.5.1803-1806.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Witkowski B, Amaratunga C, Khim N, Sreng S, Chim P, Kim S, Lim P, Mao S, Sopha C, Sam B, Anderson JM, Duong S, Chuor CM, Taylor WR, Suon S, Mercereau-Puijalon O, Fairhurst RM, Menard D. 2013. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect Dis 13:1043–1049. doi: 10.1016/S1473-3099(13)70252-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Witkowski B, Khim N, Chim P, Kim S, Ke S, Kloeung N, Chy S, Duong S, Leang R, Ringwald P, Dondorp AM, Tripura R, Benoit-Vical F, Berry A, Gorgette O, Ariey F, Barale JC, Mercereau-Puijalon O, Menard D. 2013. Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia. Antimicrob Agents Chemother 57:914–923. doi: 10.1128/AAC.01868-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Bacon DJ, Latour C, Lucas C, Colina O, Ringwald P, Picot S. 2007. 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. Antimicrob Agents Chemother 51:1172–1178. doi: 10.1128/AAC.01313-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Akala HM, Eyase FL, Cheruiyot AC, Omondi AA, Ogutu BR, Waters NC, Johnson JD, Polhemus ME, Schnabel DC, Walsh DS. 2011. Antimalarial drug susceptibility profile of western Kenya Plasmodium falciparum field isolates determined by a SYBR green I in vitro assay and molecular analysis. Am J Trop Med Hyg 85:34–41. doi: 10.4269/ajtmh.2011.10-0674. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Rason MA, Randriantsoa T, Andrianantenaina H, Ratsimbasoa A, Menard D. 2008. Performance and reliability of the SYBR green I based assay for the routine monitoring of susceptibility of Plasmodium falciparum clinical isolates. Trans R Soc Trop Med Hyg 102:346–351. doi: 10.1016/j.trstmh.2008.01.021. [DOI] [PubMed] [Google Scholar]
- 14.Mwai L, Kiara SM, Abdirahman A, Pole L, Rippert A, Diriye A, Bull P, Marsh K, Borrmann S, Nzila A. 2009. In vitro activities of piperaquine, lumefantrine, and dihydroartemisinin in Kenyan Plasmodium falciparum isolates and polymorphisms in pfcrt and pfmdr1. Antimicrob Agents Chemother 53:5069–5073. doi: 10.1128/AAC.00638-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Chaorattanakawee S, Tyner SD, Lon C, Yingyuen K, Ruttvisutinunt W, Sundrakes S, Sai-gnam P, Johnson JD, Walsh DS, Saunders DL, Lanteri CA. 2013. Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia. Malar J 12:239. doi: 10.1186/1475-2875-12-239. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Vossen MG, Pferschy S, Chiba P, Noedl H. 2009. The SYBR green I malaria drug sensitivity assay: performance in low parasitemia samples. Am J Trop Med Hyg 82:398–401. doi: 10.4269/ajtmh.2010.09-0417. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Moneriz C, Marín-García P, Bautista JM, Diez A, Puyet A. 2009. Haemoglobin interference and increased sensitivity of fluorimetric assays for quantification of low-parasitaemia Plasmodium infected erythrocytes. Malar J 8:279. doi: 10.1186/1475-2875-8-279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Lindblade KA, Steinhardt L, Samuels A, Kachur SP, Slutsker L. 2013. The silent threat: asymptomatic parasitemia and malaria transmission. Expert Rev Anti Infect Ther 11:623–639. doi: 10.1586/eri.13.45. [DOI] [PubMed] [Google Scholar]
- 19.Trampuz A, Jereb M, Muzlovic I, Prabhu RM. 2003. Clinical review: severe malaria. Crit Care 7:315–323. doi: 10.1186/cc2183. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Johnson JD, Dennull RA, Gerena L, Lopez-Sanchez M, Roncal NE, Waters NC. 2007. Assessment and continued validation of the malaria SYBR green I-based fluorescence assay for use in malaria drug screening. Antimicrob Agents Chemother 51:1926–1933. doi: 10.1128/AAC.01607-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Plouffe D, Brinker A, McNamara C, Henson K, Kato N, Kuhen K, Nagle A, Adrian F, Matzen JT, Anderson P, Nam TG, Gray NS, Chatterjee A, Janes J, Yan SF, Trager R, Caldwell JS, Schultz PG, Zhou Y, Winzeler EA. 2008. In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen. Proc Natl Acad Sci U S A 105:9059–9064. doi: 10.1073/pnas.0802982105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.WWARN. 2011. In vitroP. falciparum drug sensitivity assay using SYBR green I V1.0 procedure. Worldwide Antimalarial Resistance Network; http://www.wwarn.org/sites/default/files/attachments/procedures/INV08_PFalciparumDrugSensitivity.pdf. [Google Scholar]
- 23.Quashie NB, de Koning HP, Ranford-Cartwright LC. 2006. An improved and highly sensitive microfluorimetric method for assessing susceptibility of Plasmodium falciparum to antimalarial drugs in vitro. Malar J 5:95. doi: 10.1186/1475-2875-5-95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Chaorattanakawee S, Tyner S, Lon C, Yingyuen K, Ruttvisutinunt W, Sundrakes S, Sai-gnam P, Johnson J, Walsh D, Saunders D, Lanteri CA. 2013. Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia. Malar J 12:239. doi: 10.1186/1475-2875-12-239. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Eyase FL, Akala HM, Ingasia L, Cheruiyot A, Omondi A, Okudo C, Juma D, Yeda R, Andagalu B, Wanja E, Kamau E, Schnabel D, Bulimo W, Waters NC, Walsh DS, Johnson JD. 2013. The role of Pfmdr1 and Pfcrt in changing chloroquine, amodiaquine, mefloquine and lumefantrine susceptibility in western-Kenya P. falciparum samples during 2008–2011. PLoS One 13:0064299. doi: 10.1371/journal.pone.0064299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Zhang JH, Chung TD, Oldenburg KR. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73. doi: 10.1177/108705719900400206. [DOI] [PubMed] [Google Scholar]
- 27.Brook I. 1989. Inoculum effect. Rev Infect Dis 11:361–368. doi: 10.1093/clinids/11.3.361. [DOI] [PubMed] [Google Scholar]
- 28.Hay SI, Guerra CA, Gething PW, Patil AP, Tatem AJ, Noor AM, Kabaria CW, Manh BH, Elyazar IR, Brooker S, Smith DL, Moyeed RA, Snow RW. 2009. A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med 6:e1000048. doi: 10.1371/journal.pmed.1000048. [DOI] [PMC free article] [PubMed] [Google Scholar]
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