Pone.0004569 1.13

High Resistance of Plasmodium falciparum toSulphadoxine/Pyrimethamine in Northern Tanzania andthe Emergence of dhps Resistance Mutation at Codon581 Samwel Gesase1, Roly D. Gosling2*, Ramadhan Hashim1, Rosalynn Ord2, Inbarani Naidoo2,3, Rashid Madebe1, Jacklin F. Mosha4, Angel Joho1,4, Victor Mandia1, Hedwiga Mrema1, Ephraim Mapunda1, Zacharia Savael1, Martha Lemnge1, Frank W. Mosha4, Brian Greenwood2, Cally Roper2, Daniel 1 National Institute for Medical Research, Tanga Centre, Tanga, Tanzania, 2 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom, 3 Malaria Research Lead Programme, Medical Research Council, Durban, South Africa, 4 Kilimanjaro Christian Medical College, Moshi, Background: Sulphadoxine-pyrimethamine (SP) a widely used treatment for uncomplicated malaria and recommended forintermittent preventive treatment of malaria in pregnancy, is being investigated for intermittent preventive treatment ofmalaria in infants (IPTi). High levels of drug resistance to SP have been reported from north-eastern Tanzania associated withmutations in parasite genes. This study compared the in vivo efficacy of SP in symptomatic 6–59 month children withuncomplicated malaria and in asymptomatic 2–10 month old infants.
Methodology and Principal Findings: An open label single arm (SP) standard 28 day in vivo WHO antimalarial efficacyprotocol was used in 6 to 59 months old symptomatic children and a modified protocol used in 2 to 10 months oldasymptomatic infants. Enrolment was stopped early (87 in the symptomatic and 25 in the asymptomatic studies) due to thehigh failure rate. Molecular markers were examined for recrudescence, re-infection and markers of drug resistance and areview of literature of studies looking for the 581G dhps mutation was carried out. In symptomatic children PCR-correctedearly treatment failure was 38.8% (95% CI 26.8–50.8) and total failures by day 28 were 82.2% (95% CI 72.5–92.0). There wasno significant difference in treatment failures between asymptomatic and symptomatic children. 96% of samples carriedparasites with mutations at codons 51, 59 and 108 in the dhfr gene and 63% carried a double mutation at codons 437 and540. 55% carried a third mutation with the addition of a mutation at codon 581 in the dhps gene. This triple: triple haplotypemaybe associated with earlier treatment failure.
Conclusion: In northern Tanzania SP is a failed drug for treatment and its utility for prophylaxis is doubtful. The study founda new combination of parasite mutations that maybe associated with increased and earlier failure.
Trial Registration: ClinicalTrials.gov NCT00361114 Citation: Gesase S, Gosling RD, Hashim R, Ord R, Naidoo I, et al. (2009) High Resistance of Plasmodium falciparum to Sulphadoxine/Pyrimethamine in NorthernTanzania and the Emergence of dhps Resistance Mutation at Codon 581. PLoS ONE 4(2): e4569. doi:10.1371/journal.pone.0004569 Editor: Aric Gregson, University of California Los Angeles, United States of America Received August 1, 2008; Accepted December 12, 2008; Published February 24, 2009 Copyright: ß 2009 Gesase et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Bill and Melinda Gates Foundation through a grant awarded to the IPTi Consortiums Drug Resistance Working Group.
Grant number:38773. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] Intermittent Preventive Treatment of malaria in infants (IPTi)[2].
SP has been used in North-eastern Tanzania since the early Sulphadoxine-pyrimethamine (SP) is one of the most widely nineteen nineties[3] and was adopted as the first line antimalarial used antimalarials worldwide. It is used as first line treatment for for uncomplicated malaria nationally in 2001[4]. Plasmodium uncomplicated malaria alone or in combination with other falciparum resistance to SP has been recorded in Muheza, north- antimalarials, although it has been replaced with other antima- eastern Tanzania since 1994[5,6] and 1995[7] and when SP was larials in Southeast Asia and sub-Sahran Africa because of high adopted as the first line drug the adequate clinical and levels of resistance. SP is also recommended for use as Intermittent parasitological response (ACPR) by day 14 had already fallen to Preventive Treatment of malaria in pregnancy (IPTp)[1] in sub- 76% in Tanga region, Tanzania[8]. As part of an ongoing trial of Saharan Africa and is currently being investigated for use as IPTi using SP, mefloquine and chlorproguanil-dapsone in this PLoS ONE www.plosone.org February 2009 Volume 4 Issue 2 e4569 dhps 581 G Mutation region, we evaluated the in vivo efficacy of SP in clearing parasites (clinical efficacy) in order to understand the relationship between Symptomatic study in children aged 6–59 months.
the clinical efficacy and the protective efficacy of SP-IPTi children between the ages 6 and 59 months who attended Hale Traditionally, antimalarial drug efficacy is measured using the Health Centre during July–August 2006 with a fever or history of standard WHO 28 day in vivo protocol[9] in symptomatic, 6– fever during the study period were screened for malaria using a 59 month old children. However, results obtained in this rapid diagnostic test (RDT) (Paracheck, Orchid Biomedical population may not be representative of the efficacy of a drug Systems, Verna, India). Children with a positive RDT had a when used for prevention. When used for prevention, antimalar- thick blood smear read and those with a positive blood smear were ials work both by preventing new blood stage infections, the referred to the study clinician. Study inclusion criteria were: (1) prophylactic effect, as well as by clearing parasites present in those weight of $4.5 kgs, (2) not -enrolled in the IPTi trial, (3) absence who are asymptomatic but infected with malaria[10]. Since of severe malnutrition (weight-for-height ,3 standard deviations asymptomatic subjects generally have a lower level of parasitaemia from the norm), (4) slide-confirmed infection with P. falciparum only than clinical cases[11] even failing antimalarials may be effective with an initial parasite density of between 2,000 and 200,000 at clearing parasitaemia in asymptomatic subjects as these asexual parasites per microliter, (5) absence of general danger signs individuals are likely to have some degree of naturally acquired (inability to drink or breastfeed; vomiting; recent history of immunity. Therefore, we have studied the in vivo efficacy of SP in convulsions; lethargy or unconsciousness; inability to sit or stand both symptomatic 6–59 month old children and asymptomatic 2– up) or other signs of severe and complicated falciparum malaria 10 month old children, the target group for IPTi.
according to WHO definitions, (6) measured axillary temperature Parasite susceptibility to SP is influenced by mutations in two $37.5uC, (7) ability to attend stipulated follow-up visits, (8) genes. Resistance to pyrimethamine is determined by point informed consent provided by parent/guardian; (9) absence of mutations at codons 16, 50, 51, 59, 108 and 164 of the dhfr history of hypersensitivity reactions to SP and (10) no prior gene[12,13] and resistance to sulphadoxine by mutations at antimalarial use in the preceding 2 weeks.
codons 436, 437, 540, 581 and 613 of the dhps gene[14,15]. In Asymptomatic study in children aged between 2 and Africa, the presence of three dhfr mutations (N51I, C59R, S108N) Consent was obtained from caretakers of 2– together with two dhps mutations (A437G, K540E) prior to 10 month old infants who attended the Maternal Child Health treatment is a significant predictor of SP P. falciparum treatment (MCH) clinic for immunization or weighing, for screening for P.
failure[16,17,18]. A mutation at codon 164 in the dhfr gene falciparum infection. From those consented for screening, finger became established in south east Asia twenty years ago and was prick blood was obtained for the rapid diagnostic test, thick and found to be associated with a high level SP resistance, as well as thin blood smear preparation and filter paper samples for resistance to chlorproguanil-dapsone[19] and artesunate-dapsone- molecular studies. Children who had a positive blood slide were proguanil[20]. 164 mutants have recently begun to emerge in further assessed for their eligibility for inclusion into the study and Africa although it is not yet clear that these mutations herald the enrolled in the drug sensitivity study after obtaining an informed consent. The eligibility criteria were the same as for the study of vivo[21,22,23,24]. Because of the association of mutations in the symptomatic children except that there was no history of fever in dhfr and dhps genes with resistance to SP we studied, molecular the last 48 hours, that measured axillary temperature should be markers in symptomatic children and asymptomatic infants who less than 37.5uC and that the presence of P. falciparum parasitaemiaat any density was acceptable.
had parasitological failures.
Sample size.
In order to detect a 15% difference in adequate parasite clearance by day 28 between symptomatic 6–59 month old children and asymptomatic 2–10 month old children with80% power at the 5% significance level using a ratio of 2 The protocol for this trial and supporting CONSORT checklist symptomatic cases to 1 asymptomatic case, we estimated that 292 are available as supporting information; see Protocol S1 and symptomatic children and 146 asymptomatic infants would be Checklist S1.
Children were treated with SP (FansidarH, Roche, France) by weight (1 tablet containing The study was conducted in Hale Health Centre, Tanga 500 mg sulfadoxine and 25 mg pyrimethamine; K tablet for Region, situated 32 km north of Muheza where SP resistance was weights 4.5–10 kg, 1 tablet for 11–20 kg, 1K tablets for 21– first observed in Tanzania. The district experiences perennial, 30 kg). The content and solubility of the SP tablets were confirmed holoendemic malaria although, in recent years, transmission by solubility testing and high performance liquid chromatography appears to have declined substantially. The entomological at the London School of Hygiene and Tropical Medicine. The inoculation rate from the neighbouring district of Muheza was study drugs achieved the expected concentrations of SP in solution 148 infectious bites per person per year in 2000[25]. The study site when compared to controls (FansidarH, Roche, France) purchased was chosen due to its proximity to the site of a clinical trial of IPTi in the UK. SP was given under observation by study staff and comparing SP, chlorproguanil-dapsone and mefloquine. The children were observed for at least 1 hour after treatment. If a study protocol was approved by the Ethics Review Committees child vomited within 30 minutes of receiving the drug, the full of the National Institute of Medical Research of Tanzania and the dose was repeated. if a child vomited between 30 minutes and an London School of Hygiene and Tropical Medicine and was hour, half the dose was repeated. If a study child vomited the study registered as a clinical trial with the National Institute of Health medication twice, the study child was given rescue treatment and (Clinicaltrials.gov identifier NCT00361114). The protocol includ- excluded from the study. Rescue treatment for uncomplicated ed an arm for chlorproguanil/dapsone in the 6–59 month study malaria was artemether- lumefantrine (CoartemH, Novartis, Basel, after the SP arm was completed, however it was not possible to Swizterland) and for severe malaria was parenteral quinine.
procure the drug and recently the drug has been withdrawn from Children in the symptomatic study were seen at the clinic on days 1, 2, 3, 7, 14, 21, and 28 after treatment and PLoS ONE www.plosone.org February 2009 Volume 4 Issue 2 e4569 dhps 581 G Mutation home visits were made for those who failed to report. Parents were was recorded for every probe at each locus. A sample was encouraged to bring any child who became ill between specified considered to have a single haplotype when only one sequence visits to the clinic where they were evaluated and treated by a variant was found at each locus. Where alternative sequences were study clinician thoughout the study period. Malaria blood films present in the same these were designated as a mixed genotype and filter paper samples were obtained from children in the infection. Where mixture was detected at one locus only we symptomatic group at all active and passive follow-up time points.
inferred a mixture of 2 haplotypes which varied only by that Blood samples were not collected on days 1, 2, 3, and 21 from the asymptomatic infants if the infant remained well. However, blood We tested for the presence of mutations in addition to those at samples were collected at any time if the infant became codon 436, 437, 540, 581, and 613 of dhps by direct sequencing.
PCR products were purified using ExoSAP-ITH (USB Corpora- The primary end point, parasitological failure by tion, Cleveland, Ohio, USA). Cycle sequencing was performed day 28 was defined as (1) development of danger signs or severe using Applied Biosystems BigDye V 3.1 and samples loaded on the malaria, (2) parasitaemia on Day 2 that was higher than that on ABI-3730 capillary system. Sequence reads were checked by eye Day 0, (3) parasitaemia on Day 3 $25% of the count on Day 0, (4) and edited using the Seqman (DNAstar Inc., Madison, WI, USA).
parasitaemia on or after Day 4. Failures were further divided into The presence of SNPs was confirmed by reads through both early treatment failures (within day 3 post treatment), late clinical forward and reverse strands.
failures (recorded fever plus parasitaemia from day 4 to day 28 Recrudescent and new infections were differentiated first by post treatment), and late parasitological failure (parasitaemia at typing size and sequence of the highly polymorphic repeat region day 14 or day 28 post treatment in the absence of fever). In the of MSP2[28] and then by typing repeat length polymorphism at symptomatic study children with parasitaemia on or after day 4 the PfPK2 microsatellite marker[29]. The size polymorphism of were treated with rescue treatment if they became symptomatic or PCR amplified FC27 and IC1 fragments of MSP2, was until they reached day 28 when all parasitaemic children were determined by agarose gel electrophoresis, stained with SYBRH treated. In the asymptomatic study any child on or after day 4 with Green 1 (InvitrogenTM Ltd, Paisley, UK) and scanned on a parasitaemia was treated with rescue treatment.
TYPHOON TrioH phosphoimager (GE Healthcare, Buckingham- Review of the prevalence of the A581G mutation in shire, UK). The gel image was analysed using ‘Imagequant The literature search for reports of the A581G softwareTM'(Molecular Dynamics, Foster City, CA, USA) and mutation up to October 2007 was done using the National fragment sizes were calibrated to known fragment sizes in Library of Medicine search engines, Pubmed and Medline. The HyperladderIV (BiolineTM, London, UK) which was run in following terms were included in the search queries: dhfr, dhps, duplicate on every gel. Pre- and post-treatment samples for each sulphadoxine, sulfadoxine, pyrimethamine, Fansidar, Africa, patient were compared according to sequence and size of the PCR prevalence, malaria and resistance. To be included in the amplified MSP2 fragments. Recrudescent infections were charac- review, articles had to include analysis of codon 581 of the dhps terized as having at least one identical allele present in both pre gene in isolates collected from study sites in Africa.
and post treatment samples. Matching alleles were defined as those Blood smears were stained with for which the analysis software estimated the sizes to be within 20% Giemsa for 20 minutes and read by two independent 15 bp of each other. Samples where no alleles matched in the pre microscopists for speciation, and quantification. Parasite density and post treatment were classified as new infections. Pre-and post was estimated by counting parasites against 200 White Blood Cells treatment sample pairs which were classified as having a (WBC). A blood smear was considered negative if no asexual forms recrudescent infection according to MSP2 matches were then were seen after observing 500 WBC. Discordant results (33% compared at the Pfpk2 microsatellite locus. The Pfpk2 micro- difference in quantification or positive/negative results) were read sattelite repeat was pcr amplified using the protocol described in by a third microscopist; agreement between any two micoscopists Anderson et al[29] and fragments were run on an ABi 3730 DNA and the average parasite density were deemed to be the correct analyzer (Applied Biosystems, Foster City, USA) with LIZ-500 size finding. Parasite counts were adjusted assuming a standard WBC standard and analyzed using Genemapper software (Applied of 8000 per microlitre.
Biosystems, Foster City, USA). Any pairs of pre and post treatment DNA was extracted from bloodspots dried on filter papers by samples which matched at MSP2 but did not match at PfPK2 soaking overnight in 1 mL. of 0.5% saponin-16 phosphate were re-classified as reinfections. If either pre or post sample failed buffered saline (PBS). The segment was then washed twice in to amplify, they were classified as undetermined.
1 ml of PBS and boiled for 8 min in 100 mL PCR quality water Multiplicity of infection (MoI) was assessed by examining the with 50 mL 20% chelex suspension (pH 9.5). dhfr and dhps were numbers of alleles detected at MSP2 and pfPK2. Where the PCR amplified and point mutations at codons 51, 59, 108 and 164 number of alleles at these two loci differed, the higher of the two of the dhfr gene and codons 436, 437, 540, 581, and 613 of the dhps values was used since this is the minimum number co-infecting gene were genotyped using a dotblot methodology previously genotypes which can explain the observed diversity.
described by Pearce et al[27]. The probed blots were visualised Data were double entered into an through alkaline phosphatase-catalysed breakdown of the flouro- Access (Microsoft Corps, Seattle, USA) data base and analyzed genic substrate (ECF) (GE Healthcare, Buckinghamshire, UK) and in STATA 9.0 (Stata Corps,Texas, USA). Crude and PCR- the chemifluorescent signal scanned on a TYPHOON TrioH corrected rates (excluding new infections and indeterminate PCR) Phosphoimager (GE Healthcare, Buckinghamshire, UK). The were estimated. A survival analysis[30] was carried out by stringency and specificity of the hybridisation process was censoring children at the time of a PCR-corrected new infection confirmed by inspection of a series of four controls of known or undetermined treatment failure and loss to follow up.
single genotype variant sequence. All blots with non-specifically Logistic regression was used to determine factors associated with bound probes were stripped and re-probed. A sequence variant treatment failures (cases) compared to non-failures (controls). The was considered to be present in the PCR product when the following variables identified a priori were included in the logistic intensity of signal was higher than that of the background. The regression model: age, parasite density, study population (asymp- presence, absence, and relative abundance of hybridisation signal tomatic or symptomatic) and molecular markers. In addition, any PLoS ONE www.plosone.org February 2009 Volume 4 Issue 2 e4569 dhps 581 G Mutation factor that was significant at the 10% level in the crude analysis temperature and parasite density but not in mean haemoglobin was included in the model.
concentration (Table 1).
Further survival and regression analyses were carried out in The therapeutic efficacy of SP by day 14 and 28 post treatment is order to examine the relationship with time to failure and the shown in Table 2. There was no statistically significant difference in presence of the mutation 581G in the dhps gene.
failure rates corrected by PCR for new infections betweensymptomatic and asymptomatic groups by Day 14 (39% vs 53%; p = 0.37) or Day 28 (82% vs 77%; p = 0.35). The survival analysis(Figure 2) shows there was no significant difference between PCR Response of symptomatic children and asymptomatic uncorrected results for the symptomatic and asymptomatic groups infants to treatment with SP in the time to treatment failure. When adjusted for the effect of age, One hundred and fifty, 6–59 month-old febrile children were sex and parasite density at enrolment, the risk of treatment failure screened for malaria in July–August 2006 and 87 children who was slightly higher in the symptomatic children compared to the fulfilled the inclusion criteria were enrolled (see figure 1).
asymptomatic infants but this was not statistically significant (odds Recruitment to the study arm was stopped early when the study ratio 1.2; 95% CI 0.55, 2.9; p = 0.6). Early Treatment Failures were team observed an unacceptably high failure rate and 3 children less common and Late Parasitological Failures were more common progressed to severe disease within 3 days post treatment with SP, in the asymptomatic group compared to symptomatic children including one death due to severe malarial anaemia and (table 2). There was no statistical difference between the prevalence respiratory failure. No other adverse events were reported.
of new infections in either group (23% vs 11%, symptomatic and Between October 2006 and June 2007, 926 asymptomatic 2– asymptomatic groups respectively, p = 0.6).
10 month-old infants were screened for malaria and 25 infantswith parasitaemia were enrolled into the study arm. This study was Molecular findings also stopped early due to a high failure rate with one child New infections were detected at all time periods in the study. In the progressing to severe disease within 3 days post treatment. Apart symptomatic group 4, 5 and 10 new infections were detected between from this serious adverse event no other adverse events were days 1–3, 4–14 and 15–28 respectively and in the asymptomatic reported. The trial profiles are shown in figure 1. As expected, the group 2 new infections were detected between days 4 and 14.
two study populations differed significantly in age, weight, Molecular typing revealed a high rate of multiple clone infection both Figure 1. Trial profiles. *Incomplete follow up on Day 14 (2 in symptomatic study, 1 asymptomatic study missed visit and were followed up at latertimes).
doi:10.1371/journal.pone.0004569.g001 PLoS ONE www.plosone.org February 2009 Volume 4 Issue 2 e4569 dhps 581 G Mutation Table 1. Baseline characteristics of the two study populations.
Symptomatic children (N = 87) Asymptomatic infants (N = 25) Mean age (months, SD) Mean weight (Kg, SD) Mean haemoglobin (g/dl, SD) Mean temperature (uC, SD) Temperate Range (uC) Geometric mean density (asexual forms/mL) pre and post treatment, with between 1–4 MSP2 alleles and 1–8 mutation. The 41 references which describe them are listed in pfPK2 alleles found in individual infections. Figure 3 shows the supplementary online material (References S1). In addition to this multiplicity of infection (MoI) by showing the mean number of list a map with embedded survey details and links to the original parasite clones present at enrolment, on the day of failure and the references is available online at www.drugresistancemaps.org.
mean number of clones in cases of recrudescence by week in the The occurrence of the 581G mutation is comparatively rare in study. Sixty-seven symptomatic treatment failures cases were both Tanzania (figure 5) and in Africa as a whole (figure 6). Of successfully analysed by both MSP2 and pfPK2. The mean number 4932 isolates tested in total, 96.5% carried the 581 A wild type of clones at enrolment and day of failure is constant throughout the 4 genotype. Survey sites where 581G mutations were not found are study weeks. At all times points there were multiple clones identified in the maps as black circles whereas those in which recrudescing (treatment failures) in individual subjects. The mean 581G was found are shown as red circles with the prevalence of number of clones recrudescing was greatest in the first week of the the mutation indicated.
study and declined over time but remained greater than 1 for the In Tanzania, the 581G mutation was observed in just 3 of 16 whole period. There were 5 examples where patients were found to previous surveys. Our study in Hale found 581G in 54% of isolates have between 5–6 identical pfPK2 alleles plus 2–3 MSP2 alleles (n = 84). In Ifakara[31] in 1997, 27.8% of isolates (n = 18) were which matched on enrolment and day of failure, all occurred within found to carry the mutant while Mlimba[32] and Mbeya[33] had the first week of treatment.
low prevalence of 1.6% (n = 128) and 1.2% (n = 81) respectively.
The prevalence of mutations at each codon are shown in table 3 Surveys in Butimba (n = 57), Mlimba (n = 59), Kyela (n = 67), for dhfr and table 4 for dhps in both groups and prevalence of Masasi (n = 73), Mkuzi (n = 127), reported no 581G mutations [8], haplotypes in table 5 for dhfr and dhps. There were no mutations and similarly a survey in Muheza (n = 28) [34].
found at codon 164 in the dhfr gene but 54% of parasites in the In Africa as a whole the A581G mutation has been observed in symptomatic group carried the 581 G mutation in the dhps gene on only 14 of a total of 106 surveys. The surveys that had observed enrollment day. The ratio of 581 G mutation to 581 A wild type A581G mutation span nine countries namely Ethiopia[35] 1.6% increased from enrollment day to day of failure (0.78 and 0.93 (n = 124), Ghana[36] 0.8% (n = 126), Kenya[31] 6.1% (n = 33), respectively) but this change was not statistically significant (p = 0,5).
Malawi[37] 3.4% (n = 89), Mali[31] 100% (n = 10) and 87% Direct sequencing confirmed the presence of mutations at codons (n = 8), Sudan [38]12.4% (n = 153), Tanzania (see above) and 436, 437, 540 and 581 and did not reveal any additional mutations.
Uganda[24,39,40] 11.5% (n = 122) and 45.8% (n = 72) 45% In the symptomatic group 96% of parasites carried the triple dhfr mutant haplotype (mutants at codons 51, 59 and 108) (Table 4)showing near saturation of this haplotype. As we would be unlikely to demonstrate any further selection in dhfr haplotype we did not analyseday of failure samples for these mutations in the symptomatic study.
The results of this study demonstrate clearly that use of SP for Parasites with the double mutations in the dhps gene at codon the treatment of P falciparum malaria is dangerous in the area positions 437 and 540 were found in 65% and triple mutations with where our study was done. SP had no effect on the course of illness the additional mutation at position 581 were found in 55% of samples in four cases, three in the symptomatic group and one in the collected at enrolment. Molecular data for the asymptomatic study asymptomatic group, who developed severe disease within 3 days were too few to analyze (6/25 samples amplified for the day of of post treatment. The efficacy of SP resulted in similarly poor enrolment) and were excluded from further analysis. From samples parasitologic clearance in 2–10 month old asymptomatic infants from the symptomatic study a significant trend for increased and when compared with 6–59 month old symptomatic children. The earlier failure was seen with those carrying the 581G mutation prior WHO recommends changing first line treatment when day 28 to treatment (Figure 4). However, in a regression model, factors failure rates exceed 10%[9]. In this study, early treatment failures associated with failure in the symptomatic group were presence of of symptomatic children were 38.8% and by day 28 a staggering three mutations in the dhfr gene, age ,2 years and high parasite 82% required rescue treatment. Shortly after the study was density (Table 6) and not the presence of the 581 G mutations. The stopped artemether-lumefantrine (ALU) became the first-line results of the model did not change when looking at treatment failure treatment for uncomplicated malaria in Tanzania, although SP on or before day 3 versus failure after day 3 or treatment failure on or remains widely available through private drug stores and is before day 14 versus failure after day 14 (data not shown).
frequently used.
The primary purpose of this study was to determine if SP was Distribution of the dhps position 581 mutation in Africa more efficacious in asymptomatic parasitaemic infants than in We identified 107 surveys in 59 unique geographical localities in symptomatic children even though the drug was failing as a Africa, where P. falciparum isolates have been tested for the 581G treatment for the latter group. This has been shown to be the case PLoS ONE www.plosone.org February 2009 Volume 4 Issue 2 e4569 dhps 581 G Mutation PLoS ONE www.plosone.org February 2009 Volume 4 Issue 2 e4569 dhps 581 G Mutation Figure 2. Cumulative proportion of treatment failures of Sulphadoxine-Pyrimethamine in symptomatic 6–59 month andasymptomatic 2–10 month old children with malaria.
doi:10.1371/journal.pone.0004569.g002 for pregnant women in areas of moderate SP resistance where SP difference in response to SP between symptomatic older children may still be effective for treatment or prevention even though it and asymptomatic infants contrasting with the major differences fails when used for the treatment of symptomatic children[41,42].
observed in comparisons between asymptomatic pregnant women Because the study had to be terminated prematurely on the and symptomatic children. This difference may be due to the fact grounds of the unexpectedly high failure rates in symptomatic that infants in the second half of the first year of life, in contrast to children and asymptomatic infants, it was underpowered to show a pregnant women, have less naturally acquired immunity to difference between the two study groups. Estimates of efficacy had malaria. In pregnant women, but not infants, naturally acquired confidence intervals of 10% and 20% in the symptomatic and immunity may be sufficient to clear low density infections when asymptomatic study groups respectively and the study was parasites are exposed to an only partially effective drug. Evidence powered to detect only a 30% or more difference between groups.
from high transmission sites suggests that maternal protective Nevertheless, the findings suggest that there was no major factors do protect infants against recrudescence[43,44], however, Figure 3. Comparison of Multiplicity of Infection (MoI) at enrolment and at day of failure and the mean MoI in cases ofrecrudescence by week of failure. LEGEND: 67 symptomatic treatment failures cases were successfully analysed by both MSP2 and pfPK2 andwere sorted according to week of failure post treatment. White bars show the mean multiplicity of infection (MoI) at enrolment. Grey bars show theMoI on the day of treatment failure. Black bars show the mean number of matching alleles in the pre and post treatment sample of each patient(N = 49 after exclusion of those defined as new infections).
doi:10.1371/journal.pone.0004569.g003 PLoS ONE www.plosone.org February 2009 Volume 4 Issue 2 e4569 dhps 581 G Mutation Table 3. Prevalence of mutations in dhfr gene at day of enrolment and day of failure in all study children.
Enrolment day symptomatic cases Day of failure symptomatic cases Enrolment day asymptomatic cases Day of failure asymptomatic cases how these protective factors compare to maternal immunity itself from 55% in 1999 [34] to the current situation of 18%. This is not known. Although the study split children into symptomatic change is likely to have been driven, at least in part, by the use of and asymptomatic groups the difference between these groups SP as first line treatment for malaria from 2001 to 2007. Now that may only be that the asymptomatic children were at an earlier first line treatment for malaria has changed to combination stage of the disease. In children in a moderate transmission setting therapy across most of sub- Saharan Africa, the selection pressure in Uganda approximately 50% of asymptomatic parasitaemic for resistance to SP will have been reduced. Nevertheless the rapid children progressed to clinical disease within 30 days of detec- increase in SP resistance is a concern and an alternative drug tion[45]. This group of children, however, is relevant for this study needs to be developed for all SP-based prevention programs.
as they represent the population who would be treated with IPTi.
The molecular work was limited by the number of clones in the This study examined the treatment effect of SP and did not samples and the sensitivity of the methods to detect masked clones.
address the ability for SP to prevent blood stage infection, i.e. the In the symptomatic group 4 new infections were detected in the prophylactic effect[10]. Whether SP has any efficacy in the study first 3 days post-treatment. This is a highly unlikely result and is area when used for IPTi (both treatment and prophylactic probably due to masked clones resistant to SP becoming dominant effects[10]) will be determined in an ongoing IPTi study that will post treatment, a common criticism of PCR-correction. This be reported in early 2009 (clintrial.gov identifier: NCT00158574).
problem was also likely to interfere with the interpretation of new Within 8 years the level of SP resistance in the study area has infections when comparing the symptomatic and asymptomatic increased at an alarming rate. The day 28 ACPR has decreased groups as the symptomatic group was more likely to have heavier Table 4. Prevalence of mutations in dhps gene at day of enrolment and day of failure in all study children.
Enrolment day symptomatic cases Day of failure symptomatic cases Enrolment day asymptomatic cases Day of failure asymptomatic cases PLoS ONE www.plosone.org February 2009 Volume 4 Issue 2 e4569 dhps 581 G Mutation situations, studies of molecular markers of resistance play a key Table 5. Prevalence of point mutation haplotypes in dhfr and role in measuring patterns of resistance as there is strong evidence dhps at day of enrolment in symptomatic and asymptomatic that mutations in dhfr and dhps genes are associated with P.
falciparum resistance to SP. Previous reports from Africa havedemonstrated an association between moderate levels of resistance Number of samples to SP and triple mutation in the dhfr [16,17,18] and doublemutations in the dhps genes[18] and this association was found in our study. In South East Asia the presence of high level resistance to antifolates has been associated with the presence on an additional mutation at position 164 on the dhfr gene[20]. In the face of the very high level of SP resistance found in Hale weconsidered it possible that parasites carrying this mutation had emerged in north-eastern Tanzania. However, this did not turn out to be the case and no parasites carrying the 164 mutation were Mixed infections (unable to determine haplotype) found. Instead 55% of haplotypes, when they could be Number of samples ascertained, in addition to the 437 and 540 mutations had amutation at codon 581 in the parasite dhps gene and there was some evidence that the presence of this mutation in parasites at the start of treatment was associated earlier treatment failure.
Single (436A or C only = AAKA or CAKA) Biological plausibility of this is backed by the findings that the Single (437G only = SGKA) 581G single mutant offers resistance in vitro and that similar Single (540E only = SAEA) mutations in bacteria produce resistance to sulfa drugs[14]. Thedhps triple is recognized in South America, where it is associated Single (581G only = SAKG) with SP treatment failure[46]. Parasites carrying the 581G Double (437G+540E only = SGEA) mutation are widely distributed in Tanzania (figure 5) and Triple (437G+540E+581G = SGEG) elsewhere in Africa (figure 6). The evidence of prevalence of this Mixed infections (unable to determine haplotype) mutation is patchy with few studies looking at this locus and those that have show considerable differences in prevalence between includes mixed infections when haplotypes could be ascertained (9 for dhfrand 31 for dhps).
countries. It is likely that the large drug pressure caused when SP was the first line drug for uncomplicated malaria that has driventhe rise in frequency of first the dhps double and now the more parasite densities and more clones. Thus, in the symptomatic resistant dhps triple mutation. This early failure may prove to be group a higher proportion of recrudescences would be wrongly pertinent in the case of IPTp. ter Kuile and colleagues[41] found called new infections. The number of clones detected in samples that SP IPTp had reasonable protective efficacy against low birth- was high and in many cases these represented multiple clones weight even in areas where the day 14 treatment failure rate of SP recrudescing reflecting the high transmission and high prevalence in symptomatic children was as high as 35%. However, they of resistance genotypes circulating in the study area.
observed that the duration of prophylaxis of SP had reduced from Further in vivo efficacy studies of SP are unjustifiable in areas 4 to 2 weeks. In areas where the 581 mutation is present in where the level of SP resistance is known to be high. In such conjunction with the triple dhfr and double dhps mutants, it is Figure 4. Cumulative proportions of treatment failure of Sulphadoxine-Pyrimethamine in symptomatic 6–59 month old childrenwith malaria parasites carrying or not-carrying the A581G mutation in the dhps gene at enrolment. Unadjusted effect dhps genemutation at 581, p-value = 0.012.
doi:10.1371/journal.pone.0004569.g004 PLoS ONE www.plosone.org February 2009 Volume 4 Issue 2 e4569 dhps 581 G Mutation Table 6. Association between mutations and treatment failure.
11.0 (1.8–66.9) dhps A581G mutation 0.04 (0.002–0.9) Parasite density (Asexual forms/ul) Adjusted odds ratio: logistic regression model including age, parasite density and mutations of DHFR and DHPS. Asymptomatic cases not included in model due to toofew cases with complete molecular results (6/25).
doi:10.1371/journal.pone.0004569.t006 Figure 5. Distribution of 581G mutation in Tanzania.
doi:10.1371/journal.pone.0004569.g005 PLoS ONE www.plosone.org February 2009 Volume 4 Issue 2 e4569 dhps 581 G Mutation Figure 6. Distribution of 581G mutation in Africa.
doi:10.1371/journal.pone.0004569.g006 possible that the duration of prophylaxis given by SP will be Found at: doi:10.1371/journal.pone.0004569.s002 (0.06 MB further reduced and this may affect the protective efficacy of SP preventative strategies. Further work is needed to determine the These are the 2 study protocols which were geographical spread of this mutant and if the presence of this approved by the IRBs mutation in combination with the dhfr triple and dhps double Found at: doi:10.1371/journal.pone.0004569.s003 (0.01 MB ZIP) confers increased and earlier failure of cases treated with SP.
Supporting Information The authors wish to thank the study participants, Dr Kabula and the staff References collected by systematic review to of Hale Health Centre for their dedication and support, the study team for produce maps of the prevalence of the 581 dhps Mutation in their hard work and the other on-going studies in the area (RT,SS, ENRECA, AMANET and IPTi) for their assistance in supporting clinical Found at: doi:10.1371/journal.pone.0004569.s001 (0.04 MB care. Korogwe District Council, National Institute for Medical Research, National Malaria Control Program and the IPTi Consortium for theirsupport and feedback; Martin Grobusch, Rob Newman, Andrea Egan, CONSORT Checklist Sam Mardel, Dongmei Liu, Suzanne Welsh, Lydia Kusaga, Lorenz von PLoS ONE www.plosone.org February 2009 Volume 4 Issue 2 e4569 dhps 581 G Mutation Seidlein and Ilona Carneiro for their comments and support. Lastly the Author Contributions DSMB chaired by Bill Watkins for support and guidance.
Conceived and designed the experiments: RDG BG CR DC. Performedthe experiments: SG RDG RH RO IN RM JM AJ VM HM EM ZXS CR.
Analyzed the data: SG RDG RH RO IN RM JM CR DC. Contributedreagents/materials/analysis tools: RDG. Wrote the paper: SG RDG RHRO IN RM JM AJ VM HM EM ZXS MML FM BG CR DC.
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