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MOLECULAR EPIDEMIOLOGY OF MALARIA IN CAMEROON. XX. EXPERIMENTAL STUDIES ON VARIOUS FACTORS OF IN VITRO DRUG SENSITIVITY ASSAYS USING FRESH ISOLATES OF PLASMODIUM FALCIPARUM

ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The influence of several factors on parasite growth and 50% inhibitory concentration (IC₅₀) for chloroquine was assessed. Most isolates stored at 4°C up to 72 hours grew when they were subsequently cultivated. However, parasite viability sharply decreased from 24 hours, and the mean chloroquine IC₅₀ decreased significantly (P < 0.05). There was no evidence for selection of pre-culture populations due to storage alone. The time point when ³H-hypoxanthine was added (0 versus 18 hours) had no effect on the IC₅₀ during the 42-hour incubation, but was associated with a lower IC₅₀ when ³H-hypoxanthine was added after the initial 42-hour incubation during the 72-hour incubation. An increase in 3H-hypoxanthine incorporation and chloroquine IC₅₀ was observed as the hematocrit was increased from 1.0% to 2.5%. For the same isolates, chloroquine IC₅₀ values were generally similar when the initial parasitemia was between 0.1% and 0.5% but increased at higher (>0.75%) parasitemias. Based on these results, we recommend immediate cultivation after blood collection, a 42-hour incubation period with the addition of ³H-hypoxanthine at the beginning of incubation, a 1.5% hematocrit, and an initial parasitemia 0.1-0.5%. Further studies on serum substitutes, gas mixture, and comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol.

INTRODUCTION

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Continuous in vitro culture of Plasmodium falciparum has been made possible by the pioneering works of Haynes and others¹ and Trager and Jensen.² One of the important applications of in vitro cultivation is studies on drug resistance, which is measured by determining the drug concentration at which in vitro parasite growth (i.e., maturation of young trophozoites to schizonts) is inhibited. Two major types of microtest were developed in the late 1970s, the so-called standard microtest developed by the World Health Organization (WHO), which was based on the method of Rieckmann and others,^3,4 and the isotopic microtest.⁵ The WHO microtest was designed so that in vitro assays can be performed in field conditions, but it requires a labor-intensive examination of post-culture thick blood films. The isotopic semi-automated microtest was designed for high-output drug screening, for which well-characterized P. falciparum reference clones are used to determine drug activity. The latter method has also been successfully adapted for the determination of drug response of field isolates and for monitoring the changing trends of the epidemiology of drug-resistant malaria parasites.^6–8 The two in vitro microtests do not yield comparable results due to various differences in test procedures and different end points for measuring parasite growth inhibition.⁹

In addition to these two microtests, novel in vitro drug sensitivity assays based on the quantification of malarial lactate dehydrogenase (LDH) activity and detection of specific malarial antigen, histine-rich protein 2 (HRP 2), are under development for field use.^10–12 Other assay procedures, including flow cytometry and colorimetric assay based on the incorporation of the thymidine analog bromodeoxyuridine, have also been developed but are not being widely used.^13,14 Thus, at present, malaria researchers working in the field have a choice among at least four in vitro assays for studying drug-resistant malaria parasites.

In vitro assay is one of the important laboratory tools for researchers who are seeking to establish the relationship among in vitro response of parasites, clinical and parasitologic response of patients to drug treatment, and molecular markers for drug resistance. As an increasing number of researchers join and collaborate with a growing number of regional and international research networks on antimalarial drug resistance, standardization of test procedures to allow direct comparison of results obtained by different laboratories has become a priority. Since the basic procedures involved in performing the isotopic microtest of Desjardins and others⁵ have been maintained in the novel non-isotopic microtests, except for the use of ³H-hypoxanthine as an index for parasite growth, this microtest may serve as the starting point for the standardization of in vitro drug sensitivity assays, in particular for field use. The aim of the present study was to evaluate the influence of various factors (storage period, incubation period, pulse period, hematocrit, initial parasitemia) on the growth of fresh clinical isolates, without prior adaptation to in vitro culture, and 50% inhibitory concentration (IC₅₀) so that standardization of in vitro assays can be achieved on the basis of biologic evidence.

MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients. Fresh clinical isolates of P. falciparum were collected by venipuncture (10 mL of blood) into EDTA-coated tubes from symptomatic children 12 years old and adults consulting spontaneously at the Nlongkak Catholic missionary dispensary in Yaounde, Cameroon if the following criteria were met: parasitemia 0.2%, absence of other Plasmodium species, hematocrit 20%, and denial of recent self-medication with an antimalarial drug confirmed by the Saker-Solomons urine test.¹⁵ Pregnant women and patients presenting signs and symptoms of severe and complicated malaria were excluded. The enrolled patients were treated with amodiaquine, the first-line drug for uncomplicated malaria in Cameroon. A total of 65 consecutive fresh isolates collected in 2002–2003 were used in the experiments. The study was reviewed and approved by the Cameroonian Ministry of Public Health and Cameroonian National Ethics Committee.

In vitro culture. A short-term in vitro culture of fresh isolates was performed for either a single life cycle (42 hours) or part of two life cycles (72 hours). Infected erythrocytes were washed three times with RPMI 1640 medium (buffered with 25 mM HEPES and 25 mM NaHCO₃) by centrifugation and suspended in the complete RPMI 1640 medium with 10% fetal bovine serum at a 1.0-2.5% hematocrit. Previous studies have shown that fetal bovine serum is a suitable substitute for human serum when initiating in vitro culture of P. falciparum isolates.^16,17 For most isolates tested in this study, culture was also performed with 10% non-immune type AB⁺ human serum and 0.5% Albumax II (batch no. 326580; Invitrogen Gibco, Cergy-Pontoise, France) at a 1.5% hematocrit. Since the main purpose of this study was to define the optimal factors for in vitro drug sensitivity assays, in vitro culture was performed in duplicate in 96-well culture plates. Parasite growth was assessed by adding ³H-hypoxanthine (1 µCi per well; Amersham International, Buckinghamshire, United Kingdom) to the culture medium. A control in vitro culture was performed in 24-well plates for the morphologic evaluation of parasite maturation under microscopy.

Parasite viability. Venous blood samples were transported without ice to our laboratory within 1-2 hours after collection. Several aliquots of the whole blood were immediately stored in sterile tubes either in a refrigerator set at 4°C or at room temperature (25–30°C). The remaining sample was washed with RPMI 1640 medium. Stored aliquots were washed with RPMI 1640 at 24-hour intervals from 24 to 96 hours after blood collection. The viability of a given isolate was assessed at 24-hour intervals over a four-day period by initiating in vitro culture of aliquots at a 1.5% hematocrit and measuring the quantity of ³H-hypoxanthine incorporated over a 42-hour period (i.e., ³H-hypoxanthine added at 0 hour) by stored isolates. In this study, the criterion of viability was arbitrarily set as the capacity of fresh isolates or aliquots of isolates stored for up to 96 hours to incorporate 1,000 counts per minute (cpm) of ³H-hypoxanthine during a 42-hour incubation. Relative viability of individual isolates was defined as the percentage of parasite growth (expressed as cpm) after storage, compared with that of fresh isolates immediately cultured on day 0.

³H-hypoxanthine incorporation and incubation time. After washing the day 0 infected erythrocytes, five repeated assays were performed on fresh isolates. The assays were grouped into 42-hour versus 72-hour incubations and ³H-hypoxanthine pulsing at 0 hours (i.e., at the beginning of the incubation period), 18 hours, and 42 hours. There were two assays incubated for 42 hours and pulsed with ³H-hypoxanthine at either 0 or 18 hours, and three assays incubated for 72 hours and pulsed with ³H-hypoxanthine at 0, 18, or 42 hours. For these experiments, the hematocrit was fixed at 1.5%.

In vitro drug sensitivity assay. Stock and working solutions of chloroquine sulfate (Aventis; Antony, France) were prepared in sterile water. Eleven two-fold concentrations ranging from 3.12 to 3,200 nM (final concentrations) were distributed in duplicate in 96-well culture plates and dried in a laminar flow hood. The in vitro response was determined by the isotopic microtest developed by Desjardins and others.⁵ Infected erythrocytes were suspended in the complete RPMI 1640 medium with 10% fetal bovine serum at a 1.0-2.5% hematocrit. The suspension (200 µL) was distributed into each well. Parasitemia was adjusted to 0.6% by adding fresh uninfected erythrocytes if the initial parasitemia was 1%. For some isolates with an initial parasitemia 1%, an inoculum effect was studied by adding fresh uninfected erythrocytes to obtain various parasitemias. Parasite growth was assessed by adding ³H-hypoxanthine at different times during the incubation period.

Polymerase chain reaction. The aliquots of the same isolates stored up to 96 hours were imbibed (10 µL) onto filter paper (Isocode Stix^®; Schleichler and Schuell, Ecquevilly, France) before and after a 42-hour culture. The filter papers were thoroughly dried and sealed in airtight plastic bags until analysis. For DNA extraction, filter papers were washed in 500 µL of sterile distilled water, placed in a microtube to which 50 µL of distilled water were added, and boiled at 100°C for 20 minutes. Ten microliters were used for each amplification reaction. The nested polymerase chain reaction protocol for amplifying a fragment of the merozoite surface antigen-2 gene spanning the central polymorphic region was described in our previous study.¹⁸ The amplified fragments were resolved by agarose gel electrophoresis and compared by visual inspection.

Data analysis. Based on our previous studies using 10% human serum,^19,20 the arbitrary cut-off for in vitro chloroquine resistance was fixed at an IC₅₀ 100 nM for the purpose of this study. The actual threshold value is slightly higher since the use of fetal bovine serum results in higher chloroquine IC₅₀s, compared with human serum.17 Parasite viability expressed as the quantity of incorporated ³H-hypoxanthine and the mean logarithmic IC₅₀s obtained from the same isolates conserved at 4°C and determined at 24-hour intervals (up to 72 hours; values at 96 hours were excluded due to small sample size) were compared by repeated measures one-way analysis of variance and Dunnett’s multiple comparison post test. Data from isolates conserved at room temperature were excluded from statistical analysis due to either inadequate sample size or wide dispersion. The quantity of ³H-hypoxan-thine incorporated by the parasites and geometric mean IC₅₀s determined during different incubation periods were compared by either a paired t-test or repeated measures one-way analysis of variance and the Tukey-Kramer multiple comparison post test. The significance level was set at 0.05 for all statistical tests.

RESULTS

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Parasite viability and selection. The first set of 32 consecutive isolates (mean initial parasitemia = 1.8%, range = 0.2-18%) collected over a seven-week period were used for viability experiments. Seven isolates had initial parasitemias 1.0%, requiring dilution with uninfected erythrocytes for drug sensitivity assays. For viability experiments, the initial parasitemia of the stored aliquots of these seven isolates was also adjusted as the day 0 samples. Growth over a 42-hour period was satisfactory (mean ³H-hypoxanthine incorporation = 7,660 cpm, range = 1,820-20,900 cpm) in all isolates that were immediately cultivated. Fresh clinical isolates that were stored at 4°C within 2 two hours after blood collection were viable until 72 hours (some until 96 hours). However, viability decreased as early as 24 hours after blood collection (P < 0.05), even when the aliquots were stored at 4°C (Figure 1). Most fresh isolates kept at room temperature (25-30°C in Yaounde) were not viable beyond 24 hours. At 24 hours, isolates presenting both extremes of parasite density (0.2% and >1.0%) tended to die off rapidly. For chloroquine-resistant isolates, a decreasing tendency (P < 0.05) of the geometric mean chloroquine IC₅₀s was observed for the same isolates over the 96-hour storage period at 4°C (Table 1).

View larger version (13K): [in this window] [in a new window]       FIGURE 1. Relative viability of Plasmodium falciparum isolates after storage at 4°C (white bars) or room temperature (hatched bars) for 24-96 hours, compared with the same fresh isolates that were immediately cultured. Results are expressed as the mean ± SD relative growth. The numbers of isolates for each experiment are shown above the bars. Experiments were not done for isolates conserved at room temperature beyond 48 hours due to poor viability shown in preliminary tests.

Incubation period, ³H-hypoxanthine incorporation, and IC₅₀. The next set of 33 consecutive fresh isolates were incubated for a total of either 42 hours or 72 hours and pulsed with ³H-hypoxanthine for varying periods. All of these isolates yielded a satisfactory in vitro growth over 42-hour and 72-hour incubation periods. The amount of ³H-hypoxanthine incorporated by the parasites did not differ significantly (P > 0.05) during the 42-hour incubation period whether it was added at the beginning of the in vitro culture or after the initial incubation period of 18 hour (arithmetic mean ± SD = 8,116 ± 3,718 cpm versus 7,564 ± 4,035 cpm, respectively). There was a statistically significant (P < 0.05) decrease in ³H-hypoxanthine incorporation during the 72-hour incubation when ³H-hypoxanthine was added after the initial 42-hour incubation (mean ± SD = 4,013 ± 4,455 cpm), compared with the addition of ³H-hypoxanthine at the beginning of the culture (6,716 ± 3,709 cpm) or after the initial 18-hour incubation (7,380 ± 4,800 cpm). Chloroquine IC₅₀s were not influenced (P > 0.05) by the time point at which ³H-hypoxanthine was added (0 hours versus 18 hours) during the 42-hour incubation (Table 2). However, when the chloroquine-resistant parasites were incubated for 72 hours, there was a significant decrease (P < 0.05) in the geometric mean ^IC50 when ³H-hypoxanthine was added after the initial 42-hour incubation, compared with the values obtained when ³H-hypoxanthine was added at the beginning of the assay or after the initial 18 hours of incubation.

View larger version (35K): [in this window] [in a new window]       FIGURE 2. 3H-hypoxanthine incorporation in fresh isolates of Plasmodium falciparum during a 42-hour incubation period in relation to hematocrit (Hct) and duration of contact with 3H-hypoxanthine (white bars = 42 hours, hatched bars = 24 hours). Results are expressed as the mean ± SD counts per minute of 24 isolates.

DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The present study demonstrates the complex interplay of various factors that influence both parasite metabolism, as globally reflected by the amount of ³H-hypoxanthine incorporated into parasite DNA and RNA, and chloroquine IC₅₀s. Our data suggest that in vitro drug assays in the field are best performed on fresh isolates immediately after collection. By analogy, in non-endemic countries where in vitro drug sensitivity profiles are studied on imported malaria, drug assays should ideally be performed at the hospital where patients are admitted for diagnosis and treatment. Any considerable delay in performing the assay, i.e., 24 hours and beyond, results in a decrease in parasite viability accompanied by decreasing chloroquine IC₅₀s, introducing an important bias and render-ing more difficult current attempts by several research groups to standardize in vitro assay methods. For field researchers collecting fresh samples far from their laboratory, it is advisable to maintain the samples on ice and have them transported to the laboratory within the same day. Venous blood collected into EDTA-coated tubes is commonly used for many hematologic and biochemical investigations, and this anticoagulant has the advantages of a lack of inhibitory action on Taq DNA polymerase, compared with heparin, and negligible volume, compared with acid citrate dextrose. To determine whether the rapid decrease in parasite viability observed in the present study is related to EDTA, the possible influence of other anticoagulants on maintaining parasite viability during storage at 4°C needs to be assessed in future studies.

For in vitro assays that measure growth inhibition of parasites exposed to drugs, and not the capacity for reinvasion after being exposed to drugs, the optimal incubation period for field isolates is probably 42–48 hours, i.e., within the first complete life cycle. Previous studies have shown that a large proportion of parasites cultivated without prior in vitro adaptation does not survive the critical period beginning from the reinvasion process of the second life cycle.¹⁶ The decrease in ³H-hypoxanthine incorporation observed in our study at 72 hours, compared with the observation at 42 hours, is in agreement with this biologic phenomenon. As for the precise incubation period within the first life cycle, 42 hours may be an acceptable compromise since the majority of the isolates in our study have either attained the mature schizont stage by this time or even initiated the reinvasion process.

Most isolates are not perfectly synchronous so that within the same isolate, some parasites are in a more advanced stage than others. Moreover, at the time of blood sample collection, the stage of individual isolates may vary considerably from young rings <6 hours old to older rings that are close to 24 hours old. Thus, some isolates may initiate schizogony as early as 18 hours after incubation. For such isolates, the addition of ³H-hypoxanthine after the initial 18-hour incubation may be too late for an optimal measurement of parasite growth. Our observation is in agreement with previous studies on synchronized culture that have shown the stage-dependent incorporation of ³H-hypoxanthine; its incorporation is minimal during the ring stage (16–20 hours), increases as mature trophozoites appear, and attains the maximal level when schizonts develop.^21–23 Moreover, since the mean parasite growth and chloroquine IC₅₀ are not affected whether ³H-hypoxanthine is added at the beginning or after the initial 18 hours of incubation, it is more convenient to add ³H-hypoxanthine at the beginning of the assay, thus avoiding an additional technical maneuver and reducing the risk of bacterial contamination during this step. For investigators studying drug actions on asynchronous culture-adapted parasites, it may be recommended to add ³H-hypoxanthine after the initial 18-hour incubation to allow mature schizonts to transform into new rings and reduce the background ³H-hypoxanthine incorporation.

The absolute number of parasites is determined by hematocrit and parasitemia. For assays that are designed for 96-well culture plates, adjustment of the hematocrit to 1.0–2.0% was associated with optimal parasite growth. A hematocrit > 2.0% resulted in a suboptimal growth, possibly due to inadequate nutrients and gas exchange or accumulation of lactic acid in the medium over the 42-hour incubation period. Thus, it may be recommended to set the hematocrit at 1.5%, as in the method described by Desjardins and others,⁵ for the standardized assay protocol designed for field isolates. Moreover, the initial parasitemia needs to be adjusted to 0.1–0.5% for isotopic assays. These results confirm the previous study by Chulay and others,²¹ who concluded that incubation time, hematocrit, and initial parasitemia must be adjusted to maintain a linear relationship between the number of parasites and ³H-hypoxanthine incorporation. Higher parasitemia resulted in higher chloroquine IC₅₀s. Chloroquine and many other antimalarial drugs accumulate in infected erythrocytes in a selective manner. Increasing the number of infected erythrocytes leads to higher drug uptake from the culture medium, ^24,25 which in turn leads to higher IC₅₀s for the same isolate.

Among the factors evaluated in the present study, some quantitative changes in assay conditions resulted in a statistically significant increase or decrease in the mean chloroquine IC₅₀. However, with the exception of a few individual isolates, the classification of the isolates did not change from resistant (i.e., chloroquine IC₅₀ 100 nM) to sensitive. Thus, for most isolates, statistically significant difference in chloroquine IC₅₀s did not imply a phenotypically important change. This study was limited to chloroquine response. Further studies are required to determine whether the factors examined in this study also influence the IC₅₀s of other antimalarial drugs to a similar extent. Since the in vitro threshold for resistance is not well established for other antimalarial drugs, various factors involved in the assays need to be standardized so that IC₅₀s from different laboratories can be directly compared in future studies.

There are a number of other factors that need to be evaluated before a standardized in vitro assay protocol based on biologic evidence can be proposed to malaria researchers. From the molecular viewpoint, one of the technical difficulties in correlating in vitro response and molecular marker for drug resistance has been the presence of multiple parasite populations within a given isolate.^26,27 Multiplicity may be associated with the presence of both wild-type and mutant molecular markers, but may be phenotypically either sensitive or resistant as determined by in vitro drug assay, possibly due to the differences in the capacity for rapid adaptation to in vitro culture conditions. Thus, there may be a selection of parasite populations during cultivation. Previous studies have shown that a winnowing process occurs during prolonged in vitro cultivation, leading to the selection of a parasite population with a particular isoenzyme and phenotype, including a shift from knobby to knobless parasites and change in drug response.^28–30 Our data suggest that this winnowing process may even occur during the first in vitro life cycle of freshly isolated parasites. Further molecular characterization of the adaptation process of different parasite populations within a given isolate is necessary to explain some discordant results between in vitro response and molecular markers.

The choice of a serum supplement is another factor that should be standardized. Our previous studies have shown that the use of non-immune human serum, autologous acute-phase serum, fetal bovine serum, Albumax II, and serum-free substitutes leads to different rates of parasite growth and yields widely different IC₅₀s.^17,31,32 Albumax II may be an ideal candidate for a standardized assay because it is manufactured at an industrial scale. However, the present study suggests that optimal growth with human serum, fetal bovine serum, or Albumax II is parasite-dependent and that some isolates grow poorly when Albumax II is used.

The present study was based on the isotopic microtest developed by Desjardins and others.⁵ The isotopic assay has several advantages over the WHO microtest, including an accurate, rapid, and objective determination of parasite growth index, existence of a large database on the in vitro response of reference P. falciparum clones to antimalarial drugs (at the Walter Reed Army Institute of Research, Washington DC), and the possibility for automation. Its disadvantages include the requirements for sophisticated and expensive equipment, shipping of radiolabeled products to endemic countries, and disposal system for radioactive wastes. These disadvantages can be overcome by novel, field-applicable, enzyme-linked immunosorbent assay-based procedures that quantify either LDH or HRP II.

More data from fresh isolates are required on the possible influence of previous intake of antimalarial drugs on the in vitro response of parasites, gas mixture of the incubator, and comparison of isotopic and non-isotopic assays. Furthermore, a standardized protocol for the preparation of drug solutions, storage conditions of pre-dosed culture plates, and software for the calculation of IC₅₀ are required before in vitro drug sensitivity assays can become a standardized investigational tool for laboratory and field researchers. Once the protocol for in vitro drug sensitivity assay is standardized, it would be possible to validate the in vitro threshold for resistance in respect to the clinical response and molecular markers.

Received July 13, 2003. Accepted for publication January 8, 2004.

Acknowledgments: I thank Sister Marie-Solange Oko, the personnel of Nlongkak Catholic missionary dispensary, and Delphine Ngo Ndombol for their aid in recruiting patients.

Financial support: This study was supported by the French Ministry of Research (Program PAL+).

Author’s address: Leonardo K. Basco, Unité de Recherche Paludologie Afro-Tropicale, Institut de Recherche pour le Développement, Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, BP 288, Yaounde, Cameroon, Telephone: 237-2-232-232 Fax: 237-2-230-061, E-mails: lkbasco{at}yahoo.fr and Leonardo.Basco{at}ibaic.u-psud.fr.

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