Use of the atmospheric generators for capnophilic bacteria Genbag-CO2 for the evaluation of in vitro Plasmodium falciparum susceptibility to standard anti-malarial drugs

Over the past 20 years, many strains of Plasmodium falciparum have become resistant to chloroquine and other anti-malarial drugs [1]. Since 2001, more than 60 countries have officially adopted artemisinin-based combination therapies (ACTs) for the treatment of falciparum malaria [2, 3]. However, clinical failures or at least longer parasite clearance times with ACT have been described in Cambodia [4‐7]. The emergence and spread of resistance to most of the anti-malarial drugs require intensive research into identifying molecular markers of resistance, as well as implementing in vitro and in vivo surveillance programmes, such as those supported by the Worldwide Antimalarial Resistance Network [8, 9].

There are basically three approaches to assess anti-malarial drug susceptibility of P. falciparum: assessment of therapeutic efficacy standardized by the World Health Organization (WHO) [10], in vitro assays and molecular markers of resistance.

In a number of laboratories surveying anti-malarial drug resistance, in vitro tests are performed using the uptake of a radiolabelled nucleic acid precursor [³H]-hypoxanthine as a marker of parasite growth [11]. Other non-radioactive methods can be used: the WHO schizont maturation test by optical microscopy (Mark III) with pre-dosed plates [12], which was based on the methods of Rieckmann et al [13] and Wernsdorfer [14], a flow cytometric analysis of propidium iodide incorporation into parasite, which permits a stage-specific evaluation of anti-malarial compounds [15], a fluorescent-based technique that uses SYBR green I which binds to DNA [16, 17], and colorimetric or enzyme-linked immunosorbent assays (ELISA) to measure histidine-rich protein II (HRP2) [18, 19] or plasmodial lactate dehydrogenase enzyme (pLDH) [20, 21].

Many factors induce high variations in P. falciparum growth and 50% inhibitory concentration (IC₅₀) values and influence the results of the chemosusceptibility tests [22], such as culture medium, initial parasitaemia, haematocrit, incubation time, time point when [³H]-hypoxanthine is added, use of serum substitutes, storage conditions of sample, delay before cultivation of samples and atmosphere (gas mixtures).

Laboratories using isotopic microtest to monitor drug resistance work at different oxygen tensions: 3% O₂ [15], 5% O₂ [18, 19], 10% O₂ [23, 24], in candle jars [20, 25] (which corresponds to approximately 17-18% O₂) and >20% O₂ [21, 22] (in CO₂ incubators). WHO recommends the use of a candle jar in their in vitro microtests (Mark III). Despite the varying culture conditions, many laboratories have adopted the same threshold for the resistance to anti-malarial compounds under different oxygen tensions. For example, our previous study has shown that the chloroquine IC₅₀ values at 10% O₂ were significantly higher than those at 21% O₂ [26]. Nevertheless, it seems that O₂ concentrations between 1% and 17.5% do not affect the IC₅₀ values of quinoline-containing anti-malarial drugs [27‐29]. In contrast, the in vitro anti-malarial activity of some antibiotics was dependent on the O₂ concentration [27].

The aim of this study was to evaluate a cultivation system in which the proper conditions of atmosphere for growing P. falciparum parasites were maintained in an air-tight sealed bag. The Genbag^® system was initially designed as atmospheric generators for capnophilic bacteria. Genbag CO2^® (BioMérieux;Marcy l'Etoile, France) was used for in vitro susceptibility test of nine standard anti-malarial drugs, and the IC₅₀s were compared to those obtained with controlled incubator conditions (5% CO₂, 10% O₂ and 85% N₂).

A total of 1,098 drug-free control wells were analysed for each experimental condition. A significant difference was observed in the tritiated hypoxanthine uptake between the two conditions with 36 strains of P. falciparum (Table 1). The mean cpm values in the control wells under the controlled incubator conditions (5% CO₂ and 10% O₂) were significantly higher than those under Genbag^® conditions (2738 vs 2282 cpm, p value < 0.0001).

Compared to Genbag^® conditions (5% CO₂ and 15% O₂), the geometric mean IC₅₀ estimated under the incubator atmospheric conditions (5% CO₂ and 10% O₂) was significantly lower for atovaquone (1.2 vs 2.1 nM, p = 0.0011) and higher for the quinolines: chloroquine (127 vs 94 nM, p < 0.0001), quinine (580 vs 439 nM, p < 0.0001), monodesethylamodiaquine (41.4 vs 31.8 nM, p < 0.0001), mefloquine (57.5 vs 49.7 nM, p = 0.0011) and lumefantrine (23.8 vs 21.2 nM, p = 0.0044) (Table 1). There was no significant difference of IC₅₀ between the two conditions for dihydroartemisinin, doxycycline and pyrimethamine.

The cpm and IC₅₀ values for each anti-malarial drug were highly and significantly correlated between the two conditions (Figure 2, 3 and 4).

Based on the thresholds of decreased susceptibility defined for the incubator atmospheric conditions (5% CO₂ and 10% O₂), the following discrepancies were observed: one of 21 strains (5%) with IC₅₀ >100 nM for chloroquine [33] was <100 nM under Genbag^® conditions; seven of 20 strains (35%) with IC₅₀ >800 nM for quinine [34] was <800 nM under Genbag^® conditions; two of 20 strains (10%) with IC₅₀ >30 nM for mefloquine [35] was <30 nM under Genbag^® conditions; four of 10 strains (40%) with IC₅₀ >80 nM for monodesethylamodiaquine [36] was <80 nM under Genbag^® conditions; and none of the 13 strains with IC₅₀ >2,000 nM for pyrimethamine [37] was < 2,000 nM under Genbag^® conditions.

To reduce this difference in terms of anti-malarial susceptibility, a specific cut-off was estimated for each drug under Genbag^® conditions by regression. The cut-off was estimated at 77 nM for chloroquine (vs 100 nM in 10% O₂), 611 nM for quinine (vs 800 nM), 30 nM for mefloquine (vs 30 nM), 61 nM for monodesethylamodiaquine (vs 80 nM) and 1729 nM for pyrimethamine (vs 2,000 nM). The cut-off was not re-estimated for drugs for which all the CI₅₀ values were below the cut-off under controlled incubation conditions. For these drugs, median values were estimated: 2.9 nM for atovaquone (vs 2.5 nM in 10% O₂), 26 nM for lumefantrine (vs 30 nM), 3 nM for dihydroartemisinin (vs 3.0 nM) and 15 μM for doxycycline (vs 15 μM).

The geometric mean ratio based on the chloroquine-susceptible reference clone 3D7 (strain IC₅₀/3D7 IC₅₀) obtained under the incubator atmospheric conditions (5% CO₂ and 10% O₂) was significantly higher for chloroquine (p < 0.0001), lumefantrine (p = 0.0119), and dihydroartemisinin (p = 0.0006), but the ratio was significantly lower for mefloquine (p < 0.0001) and doxycycline (p = 0.0002) (Table 2). The geometric mean ratio based on the chloroquine-resistant reference clone W2 (strain IC₅₀/W2 IC₅₀) obtained under the incubator atmospheric conditions (5% CO₂ and 10% O₂) was significantly higher for chloroquine (p = 0.0014), quinine (p = 0.0006), monodesethylamodiaquine (p < 0.0001), lumefantrine (p < 0.0001), pyrimethamine (p = 0.0026) and was significantly lower for dihydroartemisinin (p < 0.0001) and doxycycline (p = 0.0159) (Table 3).

The first works that assessed oxygen effects on P. falciparum asynchronous cultures had shown that microaerophilic environment allowed an optimal development of parasites [38]. Parasite growth failed under strict anaerobic conditions. Plasmodium falciparum possesses a functional mitochondrial respiratory chain with oxygen consumption [39]. It has been shown that there is some protector effect of CO₂ at high oxygen concentration [38] through the medium pH, the stability (between 7.2 and 7.45) of which is required for parasite growth [40]. The standard medium RPMI 1640, buffered with 25 mM HEPES and 25 mM NaHCO₃, was optimized to maintain the pH within the physiological range in an atmosphere containing 5% CO₂. Any modification of the CO₂ concentration alters the pH of the medium, which in turn can influence the IC₅₀ values of pH-dependent drugs, such as quinolines, but not of those that are pH-independent, such as pyrimethamine. Despite similar growth and tritium-labelled hypoxanthine incorporation rates in drug-free control wells, Shenyi He et al showed that increasing the CO₂ concentration from 2.7% to 7% (with a constant 5% O₂) resulted in significantly higher chloroquine IC₅₀ values [29]. The chloroquine-resistant K1 strain showed nine-fold greater chloroquine IC₅₀ when the CO₂ concentration was increased from 2.7 to 7% [29].

The atmospheric generators for capnophilic bacteria Genbag CO₂^® evaluated in this study release about 5% CO₂ and reduce to 15% O₂ in 30 min, according to the manufacturer's specifications (http://​www.​biomerieux-diagnostics.​com/​servlet/​srt/​bio/​clinical-diagnostics/​dynPage?​doc=​CNL_​PRD_​CPL_​G_​PRD_​CLN_​62). The stated conditions are maintained at least for 48 h. In this context, it seems that the significant differences in drug IC₅₀ values and growth level (cpm) between the two conditions, the incubator atmospheric conditions (5% CO₂ and 10% O₂) and the Genbag^® conditions (5% CO₂ and 15% O₂), are due to the difference in O₂ concentration.

The IC₅₀ values of quinoline drugs, such as chloroquine, quinine, monodesethylamodiaquine, mefloquine and lumefantrine were significantly lower at 15% O₂ (Genbag CO₂^® ) than those at 10% O₂ (the incubator atmospheric conditions). This is in agreement with the previous results that showed in 136 P. falciparum fresh isolates from Comoros that the chloroquine IC₅₀ values at 10% O₂ were significantly higher than those at 21% O₂, with the means of 173.5 nM and 121.5 nM, respectively [26]. Of particular interest among the 63 isolates that were resistant in vitro to chloroquine (IC₅₀ >100 nM) at 5% CO₂ and 10% O₂, was the observation that 17 were susceptible to chloroquine (IC₅₀ < 100 nM) at 21% O₂ [26]. In the present study, only one of 21 strains with chloroquine IC₅₀ >100 nM at 10% O₂ had IC₅₀ < 100 nM at a concentration of 15%. Some studies failed to show oxygen-dependent effects of chloroquine on P. falciparum in culture [27‐29], but in these experiments less than four strains were tested. Shenyi He et al reported that the chloroquine IC₅₀ values determined in a candle jar (2.7% CO₂ and 17.5% O₂) were similar to those determined in an incubator set at 2.7% CO₂ and 5% O₂ [27]. Lin et al observed similar chloroquine IC₅₀ values with parasites incubated in 5% CO₂ and 5% or 15% O₂ generated by an AnaeroPack system [29].

In contrast, there was no significant difference of IC₅₀ values between 10% and 15% O₂ conditions for doxycycline, dihydroartemisinin and pyrimethamine. Divo et al. reported that the anti-malarial activity of different cyclines was O₂-dependent and higher at high O₂ concentrations [27]. Nevertheless, this influence of O₂ was not evident at 48 h but was profound at 96 h. In the present study, IC₅₀ values were only determined at 42 h. The class of artemisinins contains an intramolecular peroxide bridge that is situated in the sesquiterperne lactone backbone structure. The anti-malarial potency of artemisinin was enhanced by oxygen and inhibited by oxygen radical scavengers [41, 42]. The contrast between 10 and 15% O₂ is probably not high enough to observe any difference in dihydroartemisinin IC₅₀ values.

Using the threshold values for in vitro resistance established under the controlled incubator conditions (5% CO₂ and 10% O₂), 0 - 40% of discordant results, depending on the test compounds, was obtained using the Genbag^® incubation system. The effect of gas mixture on the results of chemosusceptibility assay should lead different laboratories involved in anti-malarial resistance survey to adapt a resistance threshold for each gas mixture or to use the same conditions to perform chemosusceptibility microtests. The cut-off value was re-estimated for each drug in Genbag^® conditions. The cut-off was estimated at 77 nM for chloroquine (vs 100 nM in 10% O₂), 611 nM for quinine (vs 800 nM), 30 nM for mefloquine (vs 30 nM), 61 nM for monodesethylamodiaquine (vs 80 nM) and 1729 nM for pyrimethamine (vs 2000 nM).

To reduce the effects of O₂ on IC₅₀ values between 10 and 15% O₂, the ratios of strain IC₅₀/3D7 IC₅₀ and strain IC₅₀/W2 IC₅₀ were calculated for each condition. Nevertheless, the mean ratios were not similar at 10% and 15% O₂ for most anti-malarial drugs, and the results depended on the reference clone.

The atmospheric generators for capnophilic bacteria Genbag CO2^® is an appropriate technology that can be transferred to the field for epidemiological surveys of drug-resistant malaria. The present data suggest the importance of the gas mixture on in vitro microtest results for anti-malarial drugs and the importance of determining the microtest conditions before comparing and analysing the data from different laboratories and concluding on malaria resistance.

The authors declare that they have no competing interests.