In-Vitro Susceptibility of Plasmodium falciparum to Antimalarial Drugs in Abuja, Nigeria
I Peletiri, B Matur, N Ibecheozor, J Ihongbe, T Wakama
Keywords
antimalarial drugs, plasmodium falciparum, susceptibility
Citation
I Peletiri, B Matur, N Ibecheozor, J Ihongbe, T Wakama. In-Vitro Susceptibility of Plasmodium falciparum to Antimalarial Drugs in Abuja, Nigeria. The Internet Journal of Parasitic Diseases. 2012 Volume 5 Number 1.
Abstract
The susceptibility of
Introduction
Malaria remains an important public health concern in countries where transmission occurs regularly, as well as in areas where transmission has been largely controlled, or eliminated. Malaria is a complex disease that varies widely in epidemiology and clinical manifestation in different parts of the world. This variability is the result of factors such as the species of malaria parasites that occur in a given area, their susceptibility to commonly used or available antimalarial drugs, the distribution and efficiency of mosquito vectors, climate and other environmental conditions and the behaviour and level of acquired immunity of the exposed human populations. In particular, young children, pregnant women, and non-immune visitors to malarious areas are at greatest risk of severe or fatal illness(1).
The world’s most important infectious diseases include malaria, tuberculosis and HIV/AIDS. Together, these diseases threaten the lives and well – being of millions of people, particularly in the tropics. They place enormous burdens on national health services and impair the development of many emerging nations(2). Antimalarial drug resistance has emerged as one of the greatest challenges facing malaria control today.
The WHO(3) estimated that more than 90% of the 1.5 to 2.7 million deaths attributed to malaria each year occur in African children. This study is therefore justified owing to the fact that in-vitro susceptibility testing as an epidemiological tool for assessing baseline sensitivity and for monitoring the drug response of Plasmodium falciparum over time and place can provide background information for the development and evaluation of drug policies.
The aim of this research work is to ascertain the degree of chloroquine resistant strains of Plasmodium falciparum in our environment. The specific objective on the other hand is to generate data on in-vitro susceptibility of Plasmodium falciparum to Chloroquine, Quinine, Dihydroartemisinin, and Monodesethylamodiaquine.
Materials And Methods
Study Area
The Federal Capital Territory (FCT), Abuja is the study area.
Abuja has a daily temperature range between 20.4 – 34.7oc with an average of 27.2oc/81of in January; and between 21.9 – 29.1oc with an average of 25.6oc/78of in July(4).
Sample Sites
Three representative hospitals within the FCT – National Hospital, Abuja, Wuse
General Hospital, and the Asokoro General Hospital, was the study sites.
Collection and Processing of Pre-culture Blood Specimens
Fresh blood samples already collected from patients for the diagnosis of malaria from the three representative hospitals were used following permission by the respective medical laboratory authorities. Pre-culture thick and thin films were prepared for each blood sample.
Subject Selection
This was based on exclusion and inclusion criteria set for the research. Results obtained from the processed pre-culture thick and thin films formed the basis for the criteria.
Exclusion Criteria
Patients’ blood samples were considered inadequate for the study if microscopy results of Giemsa stained films had a falciparum parasitaemia (in a thick film) of less than two trophozoites in every high power microscope field (< 1000 parasites/µl of blood). Patients with mixed infections were also excluded from the study.
Inclusion Criteria
The patients’ blood samples were considered adequate for the study if microscopy results of Giemsa stained thick films had mono-infections with Plasmodium falciparum and asexual parasitaemias in excess of 1000 parasites, but less than 80,000 parasites per µl of blood (more than one trophozoite in every high power microscope field - > / = +++).
Counting Parasite Numbers
Parasites were counted by estimating the parasite numbers/µl of blood from the thick film. This was carried out by multiplying the average number of parasites per high power field (100 x objective) by 500. Between 10-50 fields (depending on parasitaemia) were examined to determine the average number of trophozoites per high power field (HPF). Ten fields are sufficient when the parasite density is high.
The factor of 500 was proposed by Greenwood and Armstrong (5). They calculated that 5 – 8 µl is the volume of blood required to make a satisfactory thick film and that the volume of blood in one HPF (100 x objective) of a well-prepared thick film is about 0.002µl. Therefore the number of parasites per HPF multiplied by 500 gives the estimated number of parasites/µl of blood. This method, Greenwood and Armstrong found to be more accurate and quicker than counting the parasites against white cells.
In-Vitro Susceptibility Test Procedure
Materials
1. WHO pre-dosed micro – test plates for chloroquine, quinine, dihydroartemisinin, and mododesethylamodiaquine. These were supplied by the Vector Control Research Unit, Universiti Sains Malaysia (USM) under the auspices of the Communicable Disease Surveillance and Response (CDR), WHO, 1211 Geneva, 27 Switzerland.
2. Positive patients’ sample having mono-infections with Plasmodium falciparum and asexual parasitaemias in excess of 1000 parasites, but less than 80,000 parasites per µl of blood.
RPMI – 1640 medium (product number R6504), purchased from and supplied by the Vector Control Research Unit, Universiti Sains Malaysia (USM) under the auspices of the Communicable Disease Surveillance and Response (CDR), WHO, 1211, Geneva, 27, Switzerland.
Methanol
Giemsa stain
Microscope slide with frosted end.
In-Vitro Microtest Technique
The drug susceptibility of Plasmodium falciparum was determined using the World Health Organization (WHO) standardized in-vitro microtest system (6) developed by Rieckmann et al.,(7); adapting the methodology used for cultivation of Plasmodium falciparum(8). The technique involves quantitating schizont maturation following cultivation of infected erythrocytes in plates charged with defined quantities of drug.
Drug Preparation
Pre-dosed plates of chloroquine, quinine, dihydroartesiminin, and monodesethylamodiaquine were obtained from WHO in Sains, Malaysia.
In-Vitro Microtest
Following centrifugation of infected blood samples at low speed (200 rpm) the plasma was discarded and the erythrocytes (packed cells) were washed three times in culture medium (RPMI 1640 with L-glutamine, without sodium bicarbonate) at 2000 rpm for 5 minutes. 100µl of washed infected erythrocytes was then mixed aseptically with 900µl of complete culture medium (1:10) and 50µl of the blood-medium mixture (BMM) was transferred into each well of the pre-dosed plates.
The plates were incubated at 37oc for 24-30 hours (depending on schizont maturation) using the candle jar method (5% CO2)(8). All the susceptibility assays for each drug were done in duplicate.
After incubation, the plates were allowed to stand for 30 minutes in a semi-vertical position (about 45 o inclined). The supernatant was then removed, erythrocytes re-suspended in the remaining fluid, and a thick blood film was made from each well. The blood smears were air-dried for 24 hours and stained with 3% Giemsa stain for 30 minutes. The stained thick films were examined with the oil immersion objective (100x). The number of schizonts with three or more nuclei out of 200 asexual parasites (i.e., schizonts and trophozoites) was counted. For an acceptable test, schizont maturation in the control (well A) must be 10% or more (i.e., 20 schizonts with three or more nuclei per 200 asexual parasites). The counts read in the drug wells were then expressed as a percentage of the control.
Interpretation And Reporting Of Test Reports
For data analysis, a simple evaluation from a table showing the original data and those derived after standardizing for control was used.
Schizont growth (formation) at 8 pmol/well (1.6 µmol/l) or more for chloroquine, 256 pmol (51.2µmol/l) or more for quinine, 4 pmol (0.8µmol/l) or more for monodesethylamodiaquine (amodiaquine) were considered as threshold for in vitro Plasmodium falciparum resistance(1). In other words, complete inhibition of schizont growth at 4 pmol or less for chloroquine, 128 pmol or less for quinine and 2 pmol or less for momodesethylamodiaquine were considered as threshold for in-vitro Plasmodium falciparum susceptibility.
Calculation of IC50
The IC50 (50% inhibitory concentration) represents the concentration at which 50% of the isolates were inhibited from maturing to schizonts. The IC50s for the individual isolates were determined by the linear extrapolation method described by Freese et al.,(9).
Results
A total of 22 falciparum isolates were collected and cultured, but only 18 isolates were evaluated. The reasons for discarding the other tests were contamination and failure of schizonts to mature satisfactorily.
Chloroquine
Of the 18 isolates evaluated, 16 (88.9%) were resistant with a median IC50 of 0.60 µmol/l of blood (range 0.36 – 1.32 µmol/l of blood). The 2 (11.1%) other isolates were however susceptible with a median IC50 of 0.27 µmol/l of blood. The concentrations are expressed in µmol/l of blood as the malaria parasite shows selective uptake of chloroquine.
Figure 1
NB:
pmol/well = (p=pico=10-12)pmol/well is the dose of drug in each well before inoculation of parasitized blood cells.
µmol/l = (µ=micro = 10-6)µmol/l is the concentration of drug in a litre of blood realized after the inoculation of 50 µl of Blood Medium Mixture (BMM).
Both the doses/concentrations were given by the manufacturers as engraved on the WHO pre-dosed microtitre plates.
Figure 2
Quinine
Of the 18 isolates evaluated, 13 (72.2%) were susceptible to quinine with a median IC50 of 1.8µmol/l of blood-medium mixture (BMM) (range 1.4-2.3µmol/l). 5 (27.8%) of isolates were however resistant with a median IC50 of 4.7µmol/l (range 3.2 – 6.4 µmol/l). The concentrations are expressed in µmol/l BMM as the malaria parasite does not show selective uptake of quinine.
Figure 3
pmol/well = (p=pico=10-12)mol/well is the dose of drug in each well before inoculation of parasitized blood cells.
µmol/l = (µ=micro = 10-6)µmol/l is the concentration of drug in a litre of blood realized after the inoculation of 50µl of Blood Medium Mixture (BMM).
Both the doses/concentrations were given by the manufacturers as engraved on the WHO pre-dosed microtitre plates.
Figure 4
Dihydroartemisinin
The cutoff concentrations for the determination of in-vitro resistance for artemisinin compounds have yet been established. This would be realized based on comparative in vivo and in vitro tests. However, a median IC50 of 13.5nmol/l of BMM (range of 7.8 - 26 nmol/l) was realized. The concentrations are expressed in nmol/l of BMM as the malaria parasite does not show selective uptake of artemisinin.
Figure 5
pmol/well = (p=pico=10-12)pmol/well is the dose of drug in each well before inoculation of parasitized blood cells.
nmol/l = (n=micro = 10-9)nmol/l is the concentration of drug in a litre of blood realized after the inoculation of 50µl of Blood Medium Mixture (BMM).
Both the doses/concentrations were given by the manufacturers as engraved on the WHO pre-dosed microtitre plates.
Monodesethylamodiaquine
10 (55.6%) of the 18 isolates were susceptible with a median IC50 of 0.09µmol/l of blood (range 0.08-0.10µmol/l). 2 (11.1%) of the isolates with median IC50 of 0.096µmol/l had intermediate susceptibility. The remaining 6 (33.3%) isolates with a median IC50 of 0.14µmol/l (range 0.11-0.19µmol/l) were however resistant. The concentrations are expressed in µmol/l of blood as the malaria parasites shows selective uptake of monodesethylamodiaquine.
Figure 6
pmol/well = (p=pico=10-12)mol/well is the dose of drug in each well before inoculation of parasitized blood cells.
µmol/l = (µ=micro = 10-6)µmol/l is the concentration of drug in a litre of blood realized after the inoculation of 50µl of blood.
Both the doses/concentrations were given by the manufacturers as engraved on the WHO pre-dosed microtitre plates.
Figure 7
Discussion
The resistance of Plasmodium falciparum to various antimalarial drugs has become a big concern as it poses a major obstacle to the control of malaria. This has been linked to recent increases in malaria morbidity and mortality(10).
In this study, the level of chloroquine resistance was 88.9 %. In 2003, Abdullahi et al. (11)reported a 100% chloroquine resistance in Sokoto, while Sowunmi et al.,(12) reported 81.4% chloroquine resistant strains from Ibadan. Elsewhere in Papua, Indonesia Maguire et al.,(13) reported 74% chloroquine resistant Plasmodium falciparum from that part of the world. In Cameroon, a lower but moderate chloroquine resistant rate of 60% was reported by Bascol and Ringwald(14).
Quinine, with a 72.2% susceptibility rate and moderate resistant rate of 27.8% suggests that the usefulness of the drug will not last for a long time. In 1998, Hambhanje (15) reported a 10% quinine resistance strains from Papau New Guinea.
As for dihydroartemisinin, the significance of the broad variation observed in the IC50 values (7.8 – 26 nmol/l) cannot be adequately understood because discriminative values for resistance are not well – established. In their work, Cerutti et al., 16reported a range of 0.3 – 2.63 ng/ml for arteether (artemisinin) but could not analyze it because of the reason proffered above. In the laboratory, artemisinin resistant forms have since been demonstrated(17).
In conclusion, the results of this study revealed a high level and degree of chloroquine resistance, a dwindling susceptibility of quinine, and a moderate resistance to monodesethylamodiaquine.
Drug resistance will certainly be a problem for the future, and it is unlikely that the drug manufacturers will ever produce a drug which can be described as a flawless antimalarial with no possibility of resistance. Plasmodium falciparum has demonstrated that it is extremely good at adapting to any drugs we may use against it – and there is no reason to suspect that this would be different for the future if such drugs are not to become as clinically compromised as chloroquine.
Finally, the in vitro assays for assessing antimalarial drug sensitivity have become indispensable tools for the surveillance of drug resistance and the planning of therapeutic guidelines. However, the in vivo implications of these in vitro findings are not immediately known. It therefore becomes imperative for the FMOH to periodically check the antimalarial status of Plasmodium falciparum to various drugs in our environment by concurrently using both in vivo and in vitro techniques.