Marked differences in the prevalence of chloroquine resistance between urban and rural communities in Burkina Faso

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Acta Tropica 105 (2008) 81–86

Short communication

Marked differences in the prevalence of chloroquine resistance between urban and rural communities in Burkina Faso Peter E. Meissner a,b,∗ , Germain Mandi c , Frank P. Mockenhaupt d , Steffen Witte e , Boubacar Coulibaly c , Ulrich Mansmann e,f , Claudia Frey a , Heiko Merkle g , Juergen Burhenne h , Ingeborg Walter-Sack h , Olaf M¨uller a a

Department of Tropical Hygiene and Public Health, Ruprecht-Karls-University, Heidelberg, Germany b Department of Paediatrics IV Neonatology, Ruprecht-Karls-University, Heidelberg, Germany c Nouna Health Research Centre, Nouna, Burkina Faso d Institute of Tropical Medicine, Charit´ e-University Medicine, Berlin, Germany e Institute of Medical Biometry and Informatics, Ruprecht-Karls-University, Heidelberg, Germany f Institute of Medical Informatics, Biometry and Epidemiology, University of Munich, Munich, Germany g Biochemistry Center, Ruprecht-Karls-University, Heidelberg, Germany h Department of Internal Medicine VI, Clinical Pharmacology and Pharmacoepidemiology, Ruprecht-Karls-University, Heidelberg, Germany Received 29 July 2006; received in revised form 22 July 2007; accepted 30 July 2007 Available online 15 August 2007

Abstract Background: Chloroquine (CQ) resistance has reached high levels in Africa in recent years. Little is known about variations of resistance between urban and rural areas. Objectives: To compare the rates of in vivo resistance to CQ and the prevalences of the main molecular marker for CQ resistance among young children from urban and rural areas in Burkina Faso. Methods: The current analysis used the frame of a randomized controlled trial (ISRCTN27290841) on the combination CQ–methylene blue (MB) (n = 177) compared to CQ alone (n = 45) in young children with uncomplicated malaria. We examined clinical and parasitological failure rates as well as the prevalence of the Plasmodium falciparum chloroquine resistance transporter gene (pfcrt) T76 mutation. Results: Clinical and parasitological failure rates of CQ–MB differed significantly between urban (70%) and rural areas (29%, p < 0.0001). Likewise, CQ failure rates were higher in the urban setting. Matching this pattern, pfcrt T76 was more frequently seen among parasite strains from urban areas (81%) when compared to rural ones (64%, p = 0.01). In the presence of parasites exhibiting pfcrt T76, the odds of overall clinical failure were increased to 2.6-fold ([1.33, 5.16], pLR = 0.005). CQ was detected at baseline in 21% and 2% of children from the urban and the rural study area, respectively (pChi = 0.002). Conclusion: Even within circumscribed geographical areas, CQ efficacy can vary dramatically. The differences in the prevalence of pfcrt T76 and in CQ failure rates are probably explained by a higher drug pressure in the urban area compared to the rural study area. This finding has important implications for national malaria policies. © 2007 Elsevier B.V. All rights reserved. Keywords: Chloroquine resistance; Pfcrt T76; Urban; Village; Drug pressure

1. Introduction

∗ Corresponding author at: Department of Tropical Hygiene and Public Health, Ruprecht-Karls-University, Heidelberg, Germany. Tel.: +49 6221 565035; fax: +49 6221 565035. E-mail address: [email protected] (P.E. Meissner).

0001-706X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2007.07.014

Chloroquine (CQ) is severely affected by increasing drug resistance of Plasmodium falciparum (Trap´e et al., 1998; Trap´e, 2001). Drug resistance has been associated with drug pressure, long elimination half-life, transmission intensity, parasite biomass, and population movements (Talisuna et al., 2004). In CQ-resistant P. falciparum, vacuolar drug accumulation is

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Fig. 1. Study area of the Centre de Recherche en Sant´e de Nouna in Burkina Faso.

diminished but the precise mechanism is far from being understood (Yayon et al., 1984). A key mutation in the P. falciparum chloroquine resistance transporter gene (pfcrt T76) is associated with CQ resistance (Fidock et al., 2000) and has been established as the most important molecular marker of treatment failure (Djimde et al., 2001; Arav-Boger and Shapiro, 2005). In Burkina Faso, CQ and antipyretics, often combined with traditional remedies, are the most common treatment regimens for febrile young children (M¨uller et al., 2003a). In this country, in vivo CQ resistance was first reported in 1988 and clinical failure rates in children were around 5% in the early 1990s increasing to 10% and more after 2000 (Guiguemde et al., 1994; M¨uller et al., 2003b; Sirima et al., 2003). At the time of the present study (2003), CQ was still the official first-line treatment for uncomplicated malaria in Burkina Faso. The persistence of sub-curative drug concentrations in blood following treatment is one main determinant for the development and spread of resistance (White, 2004). Recent evidence suggests that CQ resistance in Africa is due to the invasion of a single mutant allele which then has been promoted by selective drug pressure (Ariey et al., 2006). Drug pressure directly relates to drug accessibility, and both drug use and resistance have been reported to be more intense in urban than in rural areas (Plowe et al., 1996). However, this association has been weaker elsewhere

(Mockenhaupt et al., 1999; Marks et al., 2005). In Burkina Faso, we tested the hypothesis that CQ resistance is more common in urban compared to rural areas. 2. Methods 2.1. Study area and study population The trial took place in the study area of the Centre de Recherche en Sant´e de Nouna (CRSN) in north-western Burkina Faso. The urban CRSN study area consists of the district capital Nouna (population, 25,000), and the rural CRSN study area of 41 villages (population, 35,000; Fig. 1). The Nouna area is a dry orchard savanna, populated mainly by subsistence farmers of different ethnic groups. Malaria transmission is intense and highly seasonal peaking during or shortly after the rainy season from June to October. The annual entomological inoculation rate varies between 100 and 1000 (Traor´e, 2003). Health services are limited to four rural health centres and the district hospital in Nouna town. 2.2. Objectives To compare, between Nouna town and the surrounding villages, (1) in vivo resistance rates to CQ and CQ–MB, (2)

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Table 1 Baseline data by treatment group and regional provenance Urban study area (n = 169)

Age (months) Weight (kg) Hemoglobin (g/dl) Hematocrit (%) P. falciparum (␮/l) (geometric mean; min; max) Pfcrt mutant allelea (T76)

Rural study area (n = 53)

CQ (n = 34)

CQ–MB (n = 135)

CQ (n = 11)

CQ–MB (n = 42)

30.9 ± 13.2 10.6 ± 3.0 10.2 ± 1.3 27.6 ± 2.5 26,226 2800, 195400 29 (85.3%)

26.8 ± 13.5 10.1 ± 2.5 10.4 ± 1.4 29.0 ± 3.2 26,930 2000, 500000 108 (80.0%)

27.0 ± 12.8 10.4 ± 2.5 10.5 ± 1.3 28.5 ± 3.1 6,785 2500, 60000 5 (45.5%)

38.0 ± 15.7 12.3 ± 3.4 10.4 ± 1.4 30.3 ± 3.5 10,968 2000, 81000 29 (69.0%)

If not specified otherwise: mean ± standard deviation. a Comprises mixed alleles (K76/T76).

prevalences of the CQ-resistance marker pfcrt T76, (3) associations between the prevalence of pfcrt T76 and treatment failures, and (4) the prevalences of CQ in blood. 2.3. Study procedures The data for this study were taken from a hospital-based randomized controlled phase IIb trial on the combination of CQ with methylene blue (MB) (Meissner et al., 2005). In October and November 2003, 226 children were recruited from the outpatient department of the Nouna hospital and included in the trial. The study protocols were approved by the Ethics Committees, CRSN, and Medical Faculty, Heidelberg University. The main inclusion criteria were age between 6 and 59 months and uncomplicated falciparum malaria (≥37.5 ◦ C axillary temperature with ≥2.000 P. falciparum parasites/␮l blood). Forty-five children were treated with CQ and 181 with CQ–MB. Treatment outcome was categorized according to current protocols with 14 days of follow-up (WHO, 2003) into early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF), and adequate clinical and parasitological response (ACPR). Losses to follow-up and dropouts due to other reasons were considered as failures in an intention-to-treat manner. Codon 76 of pfcrt was genotyped by restriction fragment length polymorphism-PCR (PCR-RFLP) as described elsewhere (Djimde et al., 2001) after extraction of DNA from blood (Qiamp, Qiagen). Mixed alleles comprising both mutant (T76) and wildtype (K76) were considered mutant. Pre-treatment CQ blood concentrations at inclusion were measured using 100 ␮l aliquots of whole blood on filter paper: In brief, CQ was extracted from paper spots using borate buffer of pH 10 and tertiary butylmethylether. The organic extract was evaporated to dryness, reconstituted in HPLC buffer, and analyzed as described by Rengelshausen et al. (2004). The limit of quantification was 50 ng/ml. The batch-to-batch accuracy of determination was −2.9, +5.2, and +0.7% at quality control concentrations of 136, 182, and 318 ng/ml, respectively. 2.4. Statistical analysis The Chi square test (Chi) was used to compare proportions, the non-parametric Wilcoxon–Mann–Whitney test (WMW) to

compare metric or ordinal data. When possible, estimates and the corresponding 95% confidence interval are given in brackets. Treatment failures were modelled using three binary variables with a multivariate logistic regression (LR) model: treatment group (CQ, CQ–MB), resistance genotype (pfcrt T76 = resistant, K76 = wildtype), and regional provenance (Nouna town, rural area). The statistical calculation was performed with SAS release 8.02 (SAS® Institute Inc., Cary, NC, USA). 3. Results One hundred sixty-nine children were recruited from Nouna town and 53 from the villages Bourasso, Bagala, and Boron, which are about 10 km away (Fig. 1). Baseline characteristics are shown in Table 1. Children from the urban as compared to rural areas were younger (27.6 ± 13.5 months vs. 35.7 ± 15.7 months, pWMW = 0.001), had a lower weight (10.2 ± 2.6 kg vs. 11.9 ± 3.3 kg, pWMW = 0.0006), a higher parasite count (geometric means, 26787/␮l vs. 9927/␮l, p < 0.0001), and slightly lower haematocrit values (28.7 ± 3.2% vs. 29.9 ± 3.4%, pWMW = 0.02). 3.1. Prevalences of pfcrt T76 in urban and rural areas Typing of pfcrt codon 76 was successful for 222/226 (98%) samples. The overall prevalence of the pfcrt T76 mutation was 171/222 (77%). It was higher in urban Nouna (81.1%; 137/169) than in rural areas (64.2%, 34/53; OR = 2.39 [1.21, 4.73], pChi = 0.01). The proportion of mixed alleles among parasites exhibiting pfcrt T76 was higher in rural (82.4%; 28/34) than in urban areas (31.4%; 43/137; pChi < 0.0001) which held true adjusting for the observed differences in age and parasite density (OR = 7.15 [2.63; 19.44], pLR = 0.001). 3.2. Association of treatment failure with pfcrt T76 Treatment failures for both CQ and CQ–MB were increased in the presence of parasites exhibiting pfcrt T76 (Table 2), and the odds of overall treatment failure were increased threefold (Table 3). Treatment failure occurred in 41.2% (21/51), 57.7% (41/71; pChi = 0.07), and 75.0% (75/100, pChi = 0.04) of children infected with parasites exhibiting pfcrt codon 76 wild-

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Table 2 Clinical or parasitological failures within subgroups Urban

mutanta

Pfcrt (T76) Pfcrt wildtype (K76) a

Rural

CQ

CQ–MB

CQ

CQ–MB

22/29(75.9%) 3/5(60.0%)

81/108 (75.0%) 13/27 (48.1%)

3/5(60.0%) 3/6(50.0%)

10/29 (34.5%) 2/13 (15.4%)

Comprises mixed alleles (K76/T76).

Table 3 Risk factors for treatment failure among 226 children Parameter

No.

Treatment failure (%)

Univariate analysis

Multivariate analysis

OR

95% CI

p

OR

95% CI

p

Residence Rural Urban

53 169

34.0 70.4

1 4.63

2.29–9.43

<0.0001

1 4.19

2.14–8.19

<0.0001

Pfcrt codon 76 K76 (wildtype) T76 (mutant)

51 171

67.8 41.2

1 3.01

1.51–6.03

0.0006

1 2.59

1.31–509

0.006

Treatment group CQ–MB CQ

147 45

59.9 68.9

1 1.48

0.70–3.17

0.27

Pre-treatment CQ in blood Absent Present

164 31

59.8 71.0

1 1.65

0.67–4.14

0.24

Parasite density (log10 ) Age (months)

222 222

n.a. n.a.

1.98 0.99

1.17–3.35 0.97–1.01

0.01 0.14

Multivariate analysis derived from logistic regression model with stepwise backward removal of factors not associated (p > 0.05). OR, odds ratio; 95% CI, 95% confidence interval; CQ, chloroquine; MB, methylene blue; n.a., not applicable.

type allele, mixed alleles (K76/T76) and the mutant allele T76, respectively. 3.3. Association of treatment failure with regional provenance Failure rates in Nouna town (CQ 73.5%, [55.6%, 87.1%], CQ–MB 69.6% [61.1%, 77.2%]) were higher than in the rural areas (CQ 54.6% [23.4%, 83.3%], CQ–MB 28.6% [15.7%, 44.6%]) (Table 3). The odds of treatment failure among children from Nouna were increased more than four-fold (Table 3). 3.4. Association of treatment failure with pfcrt T76 in urban and rural areas There were more treatment failures than responders in patients harbouring parasites with the pfcrt T76 mutation of both rural (OR, 2.35 [0.61, 9.08) and urban (OR 3.01 [1.36, 6.66]) residence but this association was significant only for the latter and larger group.

received an antimalarial drug (CQ in 80% of cases) in the week preceding recruitment (pChi = 0.021). Baseline CQ drug levels were determined in 196/222 (88.3%) of patients. Overall, 16.3% (32/196) had CQ in blood (median, 267 ng/ml; range, 54–2430): 21.1% (30/142) of the children from Nouna town and 1.9% (1/53) of children from the villages (pChi = 0.0011). Prevalences of parasites exhibiting the pfcrt T76 resistance mutation were 96.8% (30/31) and 72.0% (118/164) in children with and without CQ in blood, respectively (pChi = 0.003). Finally, we analyzed the impact of various factors on treatment failure (Table 3). Treatment failure was not associated with age, presence of CQ in blood but with baseline parasite density. In multivariate analysis (Table 3), urban residence and pfcrt T76 were found to be independent predictors of CQ treatment failure. Keeping treatment group, age, residual CQ, and parasite density in the multivariate model did not change these results substantially (urban residence, OR = 4.26 [1.94; 9.38], pLR = 0.0003; pfcrt T76, OR = 2.93 [1.39; 6.17], pLR = 0.005). 4. Discussion

3.5. Chloroquine in blood, resistant parasites, and treatment outcome More children from Nouna town (49/169, 29.0%) compared to those living in the villages (7/53, 13.2%) reportedly had

The overall efficacy rate of 38% of CQ and CQ–MB (in a low dose MB combination) in this holoendemic area in northwestern Burkina Faso is disturbingly low, particularly when considering the relatively short follow-up period of 14 days.

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Treatment failures were more than twice as common in Nouna town than in the surrounding villages. Urban–rural differences in CQ resistance have been reported before (Plowe et al., 1996; Abdel-Muhsin et al., 2002) but the extent seen here is striking. In the CRSN villages, children were older, parasite densities were lower, CQ in blood was virtually absent, and parasites less frequently exhibited pfcrt T76, which additionally to a larger extent comprised mixed alleles as compared to Nouna town. These differences may all contribute to the lower cure rates observed in Nouna town but the most important risk factor in multivariate analysis proved to be urban residence itself, independent of pfcrt T76. One feature of the rural CRSN areas is restricted in access to drugs (M¨uller et al., 2003a, 2004), which is confirmed by the present study. Although the presence of CQ in blood at recruitment did not predict treatment outcome, it nevertheless indicates the higher drug pressure in the urban study area and may still be involved in the increased failure rates, thereby selecting parasite properties beyond pfcrt T76. Altogether, our findings suggest the importance of factors not assessed here, e.g. the level of pre-existing immunity or mutations in the pfmdr gene which may modulate the degree of CQ resistance (Mockenhaupt et al., 2005). The overall higher prevalence of the pfcrt T76 mutation in the study area (77%) as compared to the lower figures obtained in previous studies in Burkina Faso (Tinto et al., 2002; AbdelAziz et al., 2005) points to an increase of CQ resistance in this country. However, the present study shows that both CQ resistance and pfcrt T76 are subject to pronounced local variation which suggests that conclusions on temporal development of resistance need to be interpreted with caution. Beyond direct drug pressure, factors such as immune responses and possibly complexity of infection may influence the occurrence of pfcrt T76 and contribute to its local variation: in previous surveys in the CRSN area, P. falciparum isolated from adults and children exhibited pfcrt T76 in 33% and 43%, respectively (Mandi et al., 2005; Abdel-Aziz et al., 2005). Similar, age-related differences have been observed in Nigeria (Mockenhaupt et al., 1999). In conclusion, the present study documents a rapid increase of clinically relevant CQ resistance in north-western Burkina Faso. CQ resistance is now established and intense in the country and, as policy from 2006 onwards, artemisinin-based combination therapy has been implemented as first-line treatment for uncomplicated malaria (Kouyat´e et al., 2007). For the interpretation of existing and future data on CQ resistance and its molecular marker, substantial variation of both parameters within relatively small geographical areas needs to be considered.

Acknowledgements The BlueCQ study was funded by a grant of DSM Fine Chemicals, Linz, Austria and by the Deutsche Forschungsgemeinschaft (SFB 544 “Control of Tropical Infectious Diseases”) at the Ruprecht-Karls-University Heidelberg.

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