QJM Advance Access originally published online on September 26, 2005
QJM 2005 98(11):789-796; doi:10.1093/qjmed/hci121
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Pre-treatment with chloroquine and parasite chloroquine resistance in Ghanaian children with severe malaria
From the 1Kumasi Centre for Collaborative Research and 4School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana, 2Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, 3Division of Infectious Diseases, Tropical Medicine & AIDS, Academic Hospital, Amsterdam, The Netherlands
Address correspondence to Dr J.A. Evans, Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Str. 74, D-20359 Hamburg, Germany. email: evans{at}kccr.de
Received 6 September 2004 and in revised form 12 August 2005
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Background: Self-medication with anti-malarial drugs is widespread, and chloroquine (CQ) resistance is increasing. The effect of these factors on the incidence and presentation of severe malaria is uncertain.
Aim: To investigate subtype of severe malaria, duration of illness, previous CQ treatment and prevalence of Plasmodium falciparum CQ-resistance markers among children presenting with severe malaria to a teaching hospital in Ghana.
Design: Prospective clinical study.
Methods: Consecutive patients (n = 189) presenting with severe malaria were examined clinically, and blood was taken for routine haematology and malaria films. Plasma and blood cells were stored and subsequently analysed by ELISA for CQ levels (n = 168) and by PCR and restriction digest for P. falciparum chloroquine resistance transporter gene (pfcrt) mutations (n = 139).
Results: Of 47 presenting with cerebral malaria, 21 had severe anaemia and 13 respiratory distress (RDS). Twenty-nine had prostration or RDS alone, 41 severe anaemia with prostration or RDS, and 72 severe anaemia not associated with coma, prostration or RDS. Of the patients studied, 77% had CQ in their plasma, and 88% were carrying P. falciparum with a CQ-resistance genotype. Significant associations were found (i) between the CQ-resistance genotype of parasites and plasma CQ levels, (ii) between the presence of CQ in plasma and the reported duration of illness, and (iii) between the reported duration of illness and the occurrence of severe but otherwise uncomplicated anaemia.
Discussion: There was extensive prior CQ use in our patients presenting with severe malaria, and a high prevalence of parasites with the CQ-resistance genotype. CQ resistance in P. falciparum may contribute to the development of severe but otherwise uncomplicated anaemia in this setting.
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Introduction |
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The problems of widespread chloroquine (CQ) resistance of the major malaria parasite Plasmodium falciparum have been recognized and discussed in considerable detail.1 Although both hospital- and community-based studies have suggested a significant impact of CQ resistance on malaria mortality,2,3 CQ remains the most commonly used drug for the treatment of fever and uncomplicated malaria in Sub-Saharan Africa. The T76 mutation of the P. falciparum CQ resistance transporter gene (pfcrt) is a variant usually representing a CQ-resistant phenotype. The variant has been shown to be selected in vivo by CQ treatment, and its presence before treatment is strongly associated with treatment failure.4
Several studies have shown that self-medication with anti-malarial drugs is very common,5,6 but the effect on the course of the disease remains unclear.
We studied 189 children admitted to a teaching hospital in Ghana, West Africa, with severe malaria according to WHO criteria, with regard to the subtype of malaria complication, the duration of illness, evidence for previous CQ treatment and the frequency of the P. falciparum genotype associated with CQ resistance.
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Methods |
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The study was carried out in the three children's wards of the Komfo Anokye Teaching Hospital (KATH), a tertiary referral centre in Kumasi, Ghana, during the end of the high and the beginning of the low malaria season (November 2000–February 2001). At this time, the treatment policy in Ghana for malaria was chloroquine or amodiaquine as the first-line drug, with sulphadoxine-pyrimethamine or quinine as second-line treatment. Early self-treatment at home with CQ was promoted by the Ministry of Health.7 Children with severe malaria were treated according to hospital and national policy with intramuscular quinine. The Ethical Committee of the School of Medical Sciences, Kumasi, granted permission for the study.
Patients were enrolled into the study if they had clinical symptoms and signs of severe malaria, according to the criteria of the WHO,8 together with a blood film positive for asexual P. falciparum parasites.
Patients with the following symptoms and signs were included and classified as follows: (i) haemoglobin (Hb) <5 g/dl or haematocrit of <15% in the absence of coma, prostration or respiratory distress (A, severe but otherwise uncomplicated anaemia); (ii) Hb <5 g/dl or haematocrit of <15% in the presence of prostration or respiratory distress (A+); (iii) Blantyre coma score of 2 or less, including those with Hb <5 g/dl (C); prostration (the inability to sit upright in a child normally able to do so, or to drink in the case of children too young to sit) with or without respiratory distress syndrome (RDS, being the presence of either marked in-drawing of the bony structure of the lower chest wall or deep (acidotic) breathing) (P).
There was some overlap between the syndromes, in that some of the patients classified as having coma (C) had concomitant anaemia and respiratory distress (Figure 1).
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Thick and thin blood films from all patients were stained with Giemsa and counted for asexual stages of P. falciparum. In no case was either P. ovale or P. malariae reported.
Haematocrit was determined using a microcentrifuge and a Hawksley haematocrit reader card and haemoglobin was measured using a Sysmex KX-21N cell counter. In addition to the routine blood samples, 1 ml heparinized venous blood was taken, and cells and plasma were separated by centrifugation. Plasma was stored at –70° C. An equal volume of 8 M urea was added to the red-cell pellet, which was stored at 4°C for DNA analysis.
CQ levels in plasma were measured by an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies.9 The lower limit of detection was 5 ng/ml. A CQ level of >100 ng/ml was considered as being compatible with recent use of CQ in an appropriate dose.
DNA was extracted from the stored red-cell pellets, and the segment of the pfcrt gene containing the polymorphism at position 76 was amplified by PCR. The PCR conditions (primer pfcrt.F: 5'-GACGAGCGTTATAGGGAATTA-3' and pfcrt.R: 5'-ATAAAGTTGTGAGTTTAGGATG-3') were an initial denaturation, followed by 30 cycles at 94°C for 40 s, 54°C for 55 s, and 72°C for 55 s, and a final extension of 10 min. Enzymic digestion with ApoI of the resulting 420 bp fragment identified parasite strains containing the T76 variant (two fragments: 348 bp and 72 bp) and/or the K76 variant (one fragment of 420 bp). T76 represents the mutant, and K76 the wild-type variant.
A trained field worker interviewed the parents or guardians accompanying the children. The questions addressed included the duration of symptoms prior to presentation, whether they had attended another health care facility, whether they had taken medication at home, where they had obtained it, and, if CQ had been taken, in what dose and when.
Statistics
Statistical analysis used JMP 5.0.1a (SAS Institute). Proportions were compared on contingency tables by 
2 statistics, and continuous variables using the Kruskal Wallis and Mann-Whitney U tests for non-parametric data.
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Clinical subtypes of severe malaria
One hundred and eighty-nine children who fulfilled the WHO criteria for severe and complicated malaria were included in the study (Figure 1). Of these, 47 had coma (C), 21 of whom also had a haemoglobin <5 g/dl, and 13 had associated respiratory distress. Twenty nine had prostration or RDS (P), 41 had Hb <5 g/dl or haematocrit <15% associated with prostration or RDS (A+), and 72 had Hb <5 g/dl or haematocrit <15% without further complications (A). Thirteen patients (6.8%) died. The presenting clinical features, CQ levels and genotyping results are described in Figure 1 and Table 1. At the time of admission, 102 patients (54%) were febrile (>37.5°C axilla) and 145 (76.7%) had parasite counts >2500/mm3.
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History according to questionnaires
Parents or carers of 159 children completed questionnaires. Eighty-nine children (56%) had visited another health care facility prior to the teaching hospital admission. Comparing the proportions of subtypes of severe malaria, there was no difference between children with and without completed questionnaires, or between those who had previously attended another health care facility and those who had not. Children were reported as having been unwell for between 0 and 14 days (median 3) prior to admission. Children with severe but otherwise uncomplicated anaemia (A) had been unwell for a significantly longer time than those with severe malaria of the other subtypes (p = 0.0002, Figure 2).
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A total of 67 (42%) gave a history of having received CQ at home prior to attending a health care facility or the Teaching Hospital. CQ had been obtained from a variety of sources, including hospitals and health centres, pharmacy shops and local drug sellers. Of the 67 respondents who reported giving CQ to their child, only 11 (16%) had done so according to the current recommendations. Eleven reported giving more than the recommended daily dose (10 mg/kg) and 38 less than the recommended dose. Seven could not recall what dose they had given. There was no correlation between reported CQ intake and the subtype of severe malaria or the duration of illness.
Chloroquine-resistance genotypes
Genotyping of the CQ resistance transporter gene (pfcrt) performed on isolates from 139 patients revealed in 122 cases (88%) the presence of the resistant T76 variant. No samples were found which contained both pfcrt variants. There was no difference in the spectrum of presenting clinical symptoms or parasite density when comparing patients with T76 and K76 isolates (Tables 1 and 2). Plasma CQ levels were significantly higher in patients with the T76 variant, compared to those with K76 (wild type) isolates (medians 77.5 ng/ml vs. 0 ng/ml, p = 0.001, Figure 3). There was no difference in duration of symptoms when comparing patients with T76 and K76 isolates.
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Plasma chloroquine levels
Plasma CQ levels were determined in 168 patients. Of these, 51 (30%) children had CQ levels >100 ng/ml, a level consistent with recent therapeutic use, and 38 (23%) had no detectable CQ (<5 ng/ml).
Children with severe but otherwise uncomplicated anaemia (A) with CQ in plasma were reported as having been unwell for longer than children with undetectable CQ in plasma (mean number of days 4.7 vs. 3.0, p = 0.03). This was not the case for children with the other subtypes of severe malaria (A+, C and P), who had an overall significantly shorter reported duration of illness. There were no significant differences in CQ levels between the various clinical subgroups, and no difference in mortality when comparing those with CQ present in plasma and those without.
Data were also collected on an additional 36 patients who presented with symptoms and signs of severe malaria but were found to have a negative malaria blood film. Although the median CQ level was slightly higher in the patients with negative malaria films, the difference was not statistically significant (p = 0.12).
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Discussion |
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In this study, a considerable proportion of children admitted to a teaching hospital in Ghana with a diagnosis of severe malaria were pre-treated with CQ and carried CQ-resistant P. falciparum strains. CQ was present in the plasma of 84% of children in whom the T76 variant was detected, and, in these, CQ levels were significantly higher than in children with the wild-type variant K76, confirming previous findings.10
The study was carried out at a time of increasing evidence of CQ resistance in Ghana, which was first reported in 198611 and has increased since then. In 1990, studies showed highest levels of resistance in the coastal area, with a relatively low frequency in the savannah and forest zones,12 and in 1991, in the coastal area, in vivo testing showed 45% resistance to CQ, with 9% of responses classified as RIII.13 By 1998, high levels of clinical and parasitological resistance were described in the forest zone, 20.8% of children showing early clinical failure. In northern Ghana in 2000, CQ treatment failure occurred in >25% of children studied, together with 57% resistant parasitological responses.14
In studies in 2003, on asymptomatic adults in a rural area close to Kumasi (from where patients are referred to our teaching hospital) with a parasite prevalence of approximately 50%, the pfcrt K76T mutation was identified in 65.5% of the individuals and CQ was detectable in the plasma of 27.6%.15 In a neighbouring district, of 1070 healthy 3-month-old children attending vaccination clinics in Kumasi, 144 (13.5%) were positive for P. falciparum by microscopy, 63% of whom had the pfcrt T76 mutation.16
CQ was present in the plasma of some of the children with the sensitive K76 variant. Interestingly, one of them had both a high CQ level and a high parasitaemia, suggesting either very recent intake of CQ or the possibility of resistance variants other than T76 of pfcrt.
Over 50% of the children presenting with severe malaria had already been treated at another health care facility. The duration of symptoms, particularly in the severely ill children with cerebral malaria, was however very short (median 1.5 days), comparable with studies from Malawi and Zambia.17,18 Reported self-medication prior to presentation was very common, consistent with other studies,5,6 and plasma levels confirmed the reported widespread use of CQ.
Until the late 1980s, few hospital-based studies reported anaemia as a severe complication of malaria, whereas more recent studies have described significant increases in such admissions.19,20 At present in Kumasi, severe anaemia is the commonest presentation of severe malaria and, as in most countries, is a major contributor to malaria burden.21,22 In the absence of data on the pattern of severe malaria admissions in the decades prior to the spread of CQ resistance in this setting, it is not possible to conclude that CQ resistance has led to an increase in the proportion of cases of severe malaria anaemia. In this context, it may be important that the presentation of severe malaria anaemia may not be uniform. Defined as a haemoglobin of <5 g/dl, severe malaria anaemia can present either in the absence or in the presence of associated symptoms such as prostration and deep breathing—in the latter case, mortality is significantly higher.23 Children presenting in this study with severe but otherwise uncomplicated anaemia (A) were reported to have been unwell for a longer time period than children showing the other clinical sub-types. A possible interpretation of the longer duration of illness and lower mortality is that there is a different underlying disease process.
Despite the absence of historical data, the pattern of associations in our study suggests the hypothesis that CQ resistance contributes to the incidence of severe but otherwise uncomplicated anaemia. In our setting, there were significant associations: (i) between CQ-resistance genotypes of P. falciparum and plasma CQ levels; (ii) between the presence of CQ in plasma and the reported duration of illness; and (iii) between the reported duration of illness and the occurrence of severe but otherwise uncomplicated anaemia. This sequence of significant associations could be interpreted as suggesting that the children eventually presenting with severe but otherwise uncomplicated anaemia developed malaria days or weeks prior to the final hospital presentation, were given CQ but, due to the presence of CQ resistance, remained parasitaemic, which resulted in a continued decline in haemoglobin level,24 eventually leading to severe anaemia and hospital admission. If the initial illness episode had been treated successfully both clinically and parasitologically, severe anaemia might not have developed. Although acute mortality from severe but otherwise uncomplicated anaemia appears to be relatively low, the necessary blood transfusions carry significant risks, particularly of HIV and hepatitis infections. It is likely that other children are dying in the community before reaching a health-care facility, and that others have a chronic anaemia, with a negative effect on growth and intellectual development as well as increased susceptibility to other infectious diseases. In a district hospital in Kenya, in anaemic patients, the risk of dying both during hospitalization and during the 8 weeks thereafter was associated with the use of CQ.25 A controlled trial to investigate the longer-term effects of CQ use would now clearly not be possible.
The implication of this study is that previous use of CQ and CQ resistance is widespread in patients presenting to hospital, and may be contributing to the presentation of severe malaria, particularly that of severe but otherwise uncomplicated anaemia.
This study focused on the prevalence of the pfcrt T76 mutation, and the assumption that this mutation confers resistance. The variant has been shown in a series of studies to be associated with both in vivo and in vitro resistance to CQ.4,26–28 The pfcrt T76 mutation is selected in vivo by CQ treatment, and its presence before treatment is strongly associated with treatment failure. A major finding has been the universal selection of the pfcrt T76 genotype in patients in whom adequate CQ treatment failed, underlining the role of drug pressure in the development of resistance.29 The prevalence of the mutation is consistently higher than the observed rates of clinical resistance, suggesting that CQ clears some infections by parasites carrying pfcrt T76, which has also been shown in other studies,30,31 but the effect was closely related to age, reflecting gradual acquisition of partial immunity.
Studies in Uganda failed to link the overall T76 prevalence to CQ resistance, mainly because the mutant genotype was present in the majority of identified infections. The prevalence of K76 wild type varied widely however, and the ratio between T76 and K76 prevalences showed a strong correlation, with clinical and parasitological resistance with lower levels of K76 being related to higher resistance, suggesting that the disappearance of wild-type parasites may be one of the last stages in the development of high CQ resistance.32
The present study supports the current thinking that CQ monotherapy needs to be replaced with an alternative regime, which is now being done in Ghana.33 Our data indicating that only 16% of children received CQ according to the current dosage recommendations underlines the importance of combining the introduction with a public health campaign to ensure that new therapies are taken in an appropriate manner. A simple replacement with another drug will not be sufficient; it would need to be accompanied by a reorientation in the management of uncomplicated malaria by the peripheral health services.
Our study also underlines the importance of studies to monitor patterns of presentation of disease.3 Following the successful implementation of new effective drug regimes, it will be important to monitor the effect on the number of admissions of severe anaemia related to malaria.
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Acknowledgments |
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This study was supported by the German National Genome Research Network (01GS0112). The work presented forms part of the doctoral thesis of DT.
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