In vitro tests for drug resistance in Plasmodium falciparum

The contribution on drug resistance in Plasmodium falciparum in five States of India by Anvikar et al1 in this issue deserves particular attention since it underlines the importance of in vitro investigations for the clinical activity and its limitations for the efficacy of antimalarial drugs. On the limited sample from five States of India, it illustrates the lack of therapeutic activity of chloroquine, supported by restriction fragment length polymorphism analysis of pfcrt gene for the detection of K76T. Thus, chloroquine should be replaced by more effective drugs in the treatment of P. falciparum infections, wherever it is used in India. Also the record for monodesetylamodiaquine indicates reduced antimalarial activity in Orissa, Jharkand and Chhattisgarh, overlapping with resistance to chloroquine. On the contrary, the response to dihydroartesunate and to mefloquine seems to be fully preserved. By restricting the monitoring to 108 isolates of P. falciparum, the extent of the exercise seems to be well-below the critical mass, but it is a sound beginning, setting the signs for a major geographical expansion.

The creation of tests for the antimalarial activity against P. falciparum was preceded by the possibility of cultivating the parasite in vitro2. Two years later, Rieckmann et al3 applied the in vitro culture technique to parasites in continuous culture. Based on these observations, it was apparent that natural isolates of P. falciparum could also be exposed, ex vivo, to mixtures of antimalarial drugs and plasma-free culture medium and to measure, hereby, the specific activity of the antimalarial drugs. These results were assessed under the auspices of the World Health Organization which issued in the 1980s the initial versions of the “in vitro micro-test for the assessment of the response of Plasmodium falciparum to chloroquine, mefloquine, quinine, sulfadoxine/pyrimethamine, and amodiaquine”4. Since then, a number of modifications were introduced (Table). This Table also indicates additional compounds considered suitable for the application of the method to other chemical classes.

Table In vitro sensitivity micro-tests for P. falciparum

The test for P. falciparum is a schizont maturation inhibition test. It uses the natural ability of schizont formation in the intravascular environment to facilitate the reading of schizonts in the test environment and is, therefore, prone to over-breeding. Thus, particular attention has to be given to avoid over-breeding, an event leading to the cancellation of the results.

The test results are conveniently analysed by the method for the evaluation of dose-effect experiments13. The computerized programme provides an analysis of the inhibitory concentrations (ICs) and a series of regression parameters useful for comparisons with other log-probit regressions14, inter-alia the slope of the regression, the heterogeneity of results and the correlation coefficient.

The relative efficacy of antimalarial drugs is subject to wide variation, e.g. the value for the IC₉₉ of monodesethylamodiaquine is well below 500 nM, whereas that of quinine lies above 10 μM. That of fosmidomycine is even higher. The evaluation of the micro-tests is to be adjusted to the blood concentrations of the compounds. This is also the basis for the calculation of the estimated IC₅₀, IC₉₀ and IC₉₉. A unique quotation of an IC₅₀ is wrong since it reflects only 50 per cent inhibition and omits the inhibition at higher ICs which may very well be situated outside the area of sensitivity. For the evaluation of grouped data, it may also be convenient to calculate the cut-off concentration of schizont maturation mean cut-off concentration (MCOCM) as it provides an overview of the general situation.

MCOC = Sum of first concentrations with complete interruption of Schizont maturation / n

Values near the limit of activity are a signal of impending resistance

Significant incidence of P. falciparum infections in India is marked with choloroquine as well as sulphadoxine/pyrimethamine. Chloroquine as well as sulphadoxine/pyrimethamine are not anymore useful, and the search has to be concentrated on the artemisinin based combination therapy (ACT) with mefloquine and a suitable artemisinin derivative, or on lumefantrine/β-artemether. This exercise will be useful if it includes wide enough a case clientele for the selected area, i.e. one or two checking areas per State. For instance, none of the States at >90 ° East of Greenwich (Assam, etc.) was included in the analysis. In addition, these States are within easy reach of neighbouring Myanmar, a country already experiencing resistance to mefloquine and artemisinin. The situation calls for making the laboratory technicians familiar with the conduct and interpretation of the in vitro micro-test since in vivo tests are time consuming. This training should be carried out in an area with a significant incidence of P. falciparum.

Finally, it seems to be worth mentioning that also the in vitro assessment of antimalarial drugs against P. vivax has been developed to maturity15. It seems to be appropriate to India since P. vivax is the prevailing species in this country and chloroquine-resistance has reached Myanmar.