Plasmodium falciparum: higher incidence of molecular resistance markers for sulphadoxine than for pyrimethamine in kasangati, uganda

Tropical Medicine and International Health Plasmodium falciparum: higher incidence of molecular resistancemarkers for sulphadoxine than for pyrimethamine in Kasangati,Uganda 1 Department of Biochemistry, Makerere University, Kampala, Uganda2 Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden In November of 2000, Uganda changed its anti-malarial policy to replace chloroquine (CQ) with acombination of CQ and sulphadoxine–pyrimethamine (SP) as the first line agents. Information waslimited on the efficacy of either drug. The present study was designed to provide baseline informationon the efficacy of SP and the prevalence of molecular markers that are associated with SP resistance.
Blood samples were collected on filter paper from 169 consenting patients who were diagnosed withmalaria. Patients were treated with SP and followed for 14 days using the WHO clinical guidelines.
The samples were analysed for molecular resistance markers and correlation of the molecular markerswith clinical findings was assessed. SP monotherapy was efficacious for 140 of 163 (85.9%) treatedpatients. We found a high level of mutations in alleles which have previously been reported to beassociated with SP resistance, but there was no correlation between clinical outcomes and molecularmarkers. With the exception of codon S108 in dhfr (dhfr S108N was at 94.9%), frequencies ofdihydropteroate synthase (dhps) mutant and mixed alleles combined (A437G 89% and K540E 83.9%)were higher than those of dihydrofolate reductase (dhfr) (N51I 58.4%, C59R 31.3%).
keywords Plasmodium falciparum, molecular markers, dihydropteroate synthase, dihydrofolatereductase, sulphadoxine, pyrimethamine regimens and this is the way to go if spread of highly resistant parasites is to be controlled (White 1999).
Malaria is the leading cause of morbidity and mortality in Artemisinin based combinations (ACTs) are therefore Uganda, accounting for 30% of outpatient attendance and recommended in management of uncomplicated malaria in 20% of hospital admissions (Anonymous 1991). The many areas of the world, but this kind of therapy remains Uganda Malaria Control Programme of the Ministry of expensive for most sub-Saharan populations and malaria Health (MOH) recommended a policy change to a com- control programmes (Bloland et al. 2000).
bination of chloroquine (CQ) and sulphadoxine–pyri- Uganda chose a cheaper combination of CQ and SP, methamine (SP), replacing CQ as first line therapy for which had been proven to work in Tanzania and other uncomplicated malaria in December of 2000 (Kamya et al.
malaria endemic areas (Tarimo et al. 2002). There was, however, limited information on the levels of resistance in The policy change was effected as the resistance levels to Uganda, and only a few studies had documented molecular CQ reached a national average of 40% (Kamya et al.
markers associated with SP resistance (Jelinek et al.
2002). Many countries in East Africa including Malawi, 1999a,b). A review paper of all the available data on drug Kenya and Tanzania had by the late 1990s switched to SP, resistance since it was documented for CQ in 1988 was which is the next affordable alternative (Bloland et al.
published 2 years after the change of policy (Kamya et al.
1993; White 1999; Ogutu et al. 2000). In the countries where SP monotherapy was widely used, resistance devel- The molecular markers used for investigation of resist- oped quite rapidly (Bousema et al. 2003; Bwijo et al.
ance to SP are mutations in the genes coding for 2003), necessitating a need to review policy within a short dihydrofolate reductase (dhfr) and dihydropteroate syn- time. It is generally agreed that combination therapy would thase (dhps) enzymes. There are indications of a relation- deter the rate of development of resistance to individual ship between mutations in these genes and treatment Tropical Medicine and International Health H. Sendagire et al. Plasmodium falciparum and molecular resistance markers outcome (Kublin et al. 2002). Recently a study comparing Documented fever was defined as axillary temperature of SP with different combination therapies in Uganda inclu- ded molecular data that showed high frequencies ofmutations in dhfr and dhps (Dorsey et al. 2003).
The clinical study reported here was carried out in 2000 before the change of treatment policy to SP plus CQ The patients received a single dose of 25 mg/kg sulpha- became effective at end of 2000, and therefore only doxine and 1.25 mg/kg pyrimethamine (Fansidar; Roche, involved SP treatment. During this study, we assessed Basel, Switzerland). Drugs were administered under direct clinical efficacy of SP for treatment of uncomplicated observation to ensure compliance, such that if a patient malaria and also analysed P. falciparum isolates from vomited within 30 min the dose was repeated. Those who patients’ blood obtained on day 0 for the frequencies of vomited the drugs more than once were excluded from the mutations in the genes coding for dhfr and dhps. We aimed study and given either parenteral CQ or quinuine (QNN).
at establishing the baseline frequencies against which we The patients who failed treatment were administered could gauge subsequent mutational changes brought about 10 mg/kg QNN every 8 h for 7 days. Those who devel- by the adoption of SP as an ingredient of the first line oped severe malaria were referred to Mulago Hospital.
treatment. Here, we report our findings and compare themolecular markers to treatment outcome.
From patients enrolled into the study a blood sample (i.v.) was collected for confirmatory thin and thick smears, andhaemoglobin was estimated. Patients with a blood hae- moglobin level <9 g/100 ml were given ferrous sulphate The study was conducted at Kasangati Medical Complex, and, when clinical signs suggested worm infection, tested an outreach health unit of the Institute of Public Health, for stool ova and cysts. A filter paper sample was collected Makerere University and a district referral unit for Wakiso on the first day and kept for future molecular analysis district. It is 20 km north-east of Kampala and is consid- described below. The patients were asked to revisit the ered a peri-urban area. Malaria is meso-endemic in clinic on follow-up days 3, 7 and 14 plus any other day Kasangati, with peak transmission occurring after each of when they felt unwell within the follow-up period of two rainy seasons a year. The study was conducted 14 days. Follow-up consisted of history taking, a physical according to WHO criteria (WHO 1996) and in compli- examination and blood examination. On each day the ance with guidelines of ethical approval granted by Uganda recruited patient revisited the clinic, a case record form was National Council of Science and Technology (UNCST).
completed, a blood smear prepared and examined, andfour blood drops blotted on filter paper for parasite DNAanalysis. When a patient had malaria parasites in the blood, parasitemia was determined and a full hemogram Patients who presented with symptoms suggestive of carried out. Patients who failed to return were visited at malaria were screened and later considered for recruit- their homes by our social worker/health visitor and ment if they fulfilled the following inclusion criteria: brought to the health unit for examination and medical 6 months of age and above; a positive blood smear with P. falciparum mono-infection and parasite density morethan 1000 asexual parasites per microliter; uncomplicated malaria with absence of symptoms of severe malaria ordanger signs, e.g. convulsions, excessive vomiting, drow- The thick smears were stained with 10% Giemsa for siness or inability to feed; axillary temperatures of 10 min, and asexual parasites were counted against 37.5 °C and above or history of fever in the last 48 h; every 200 white blood cells (WBCs). Results were multi- absence of history of allergy to sulpha-dugs; residence plied by 40 to determine the parasite density (parasites/ll), within an accessible address and willingness to return to assuming a normal WBC count of 8000/ll.
the clinic for follow-up; provision of informed consent.
Clinical evaluation involved taking a history of the patient including antimalarial therapy within the previous 72 h.
A physical examination was carried out and the tem- DNA from cut sections of the filter papers was extracted by perature was measured using a clinical thermometer.
the chelex method as described earlier (Plowe et al. 1995).
Tropical Medicine and International Health H. Sendagire et al. Plasmodium falciparum and molecular resistance markers PCR- Restriction fragment length polymorphism (RFLP) Plasmodium falciparum resistance to SP was earlier Mutations at positions 108, 51 and 59 of the pfdhfr gene, reported (Ndyomugyenyi & Magnussen 1997) to be 2% in as well as at positions 437 and 540 of the pfdhps gene were malaria patients from Kigezi District which is a rural detected by nest PCR-RFLP methods based on previous setting of Uganda, while, presumably to due higher drug published data (Duraisingh et al. 1998; Masimirembwa use, urban areas such as Kampala city experienced 10% et al. 1999), with minor modifications.
parasite resistance to SP around year 2000 (Kamya et al.
For the analysis of the pfdhfr single nucleotide 2001). Accordingly, in order to work within 95% confid- polymorphisms (SNPs), the primers used for the primary ence limits at power of 80 and allow for 15% loss to amplification were: fw: (5¢-ATG ATG GAA CAA GTC follow-up, we based our calculations on the above levels of TGC GAC-3¢), rev: (5¢-CTT GAT AAA CAA CGG ACC resistance and recruited 182 patients for the study.
CTC-3¢). Cycling conditions were an in initial 94 °C Our outcome parameters were clinical and parasitological incubation for 45 s followed by a total of 45 cycles outcome at day 14. Clinical outcomes were classified as with denaturation at 92 °C for 30 s, annealing initially adequate clinical response (ACR) or early treatment failure at 56 °C for 30 s and elongation at 72 °C for 45 s.
(ETF) and late treatment failure (LTF). The parasitological The annealing temperature was gradually lowered to outcomes were described as sensitive (S) or resistant (RI, RII and RIII) according to the WHO clinical and parasito- For the detection of Ser108Asn a nest PCR was logical classification systems (WHO 1996; WHO Expert performed with the following primers: fw: (5¢-ACT ACA Committee on Malaria 2000) We used the chi-squares and CAT TTA GAG GTC TAG G-3¢), rev: (5¢-GGT TCT AGA Pierson’s significance test to assess for statistical significance CAA TAT ACC ATT TAT CC-3¢); for the analysis of the in correlation of clinical with molecular findings.
presence of the pfdhfr SNP coding for Asn51Ile andCys59Arg, the primers were: fw: (5¢-GCC ATA TGT GCA TGT TGT AAG GTT GAA AG-3¢), rev: (5¢-CAT ATTTTG ATT CAT ATG TTG TAA CTG CTC-3¢). Cycling While the clinical and parasitological evaluations of drug conditions for the nested reactions were 94 °C for 1 min efficacy were undertaken mostly during year 2000, setting followed by 10 cycles of 94 °C for 20 s, 60 °C for 30 s, up the molecular protocols, training personnel in DNA 72 °C for 30 s and 35 cycles of 94 °C for 20 s, 58 °C for methods and transferring the technology of genotyping 30 s, 72 °C for 30 s. A final 10 min run at 72 °C finished took about a year (i.e. late 2000 to late 2001). Actual analysis of parasite dhfr and dhps DNA was then under- The primers and conditions for pfdhps PCR were as taken in 2002 and completed by early 2003. Besides described (Duraisingh et al. 1998). For the analysis of the investigating the mutations in dhfr and dhps, we set up pfdhfr SNPs, restriction enzyme Asn108, AluI was MSP 2 genotyping procedures (Cattamanchi et al. 2003) employed for detection of Ser108; Tsp509I for Ile51and where, in 28-day follow-up studies, we analyse parasite TaqI for Arg59. For the analysis of the two pfdhps SNPs, DNA to resolve cases of parasitological resistance into AvaII was used for the detection of Gly437, and FokI for recrudescence and re-infection. We screened 712 patients the analysis of the presence of Glu540. All restriction and recruited 182 for follow-up. Of these, 12 patients were enzymes were from New England Biolabs, Beverly, MA, lost to follow-up for various reasons; seven changed USA and digestions were carried out according to the address; the home visitor could not trace five because of manufacturer’s recommendation. All restriction products poor or wrong directions given by the patients; one patient were separated through 2% agarose gel electrophoresis and was transferred to Mulago hospital after showing signs of visualised upon UV exposure. The results were compared severe malaria with convulsions. Of the remaining 169 with those of control plasmid DNA from parasite strains patients, six were excluded from the study because they 3D7, FCR3 and V1/S. Part of the molecular genotyping took non-study medication during the course of follow-up.
was performed at Uppsala University but, during the study, Characteristics for the 169 patients that were initially a laboratory equipped for PCR and restriction analysis was established at Makerere University. For the main part of The general picture from clinical and parasitological the study, the full molecular analysis was performed at outcome data (Table 2) is that SP has a good effect with Makerere University using the protocols described above.
87.5% showing ACR. This is paralleled by 86.3% of cases Several Ugandan scientists have now been trained in the showing a parasitological response of sensitivity (S).
However, screening for molecular markers of SP resistance Tropical Medicine and International Health H. Sendagire et al. Plasmodium falciparum and molecular resistance markers the treatment outcome (Table 4). Because of the largenumbers of dhps mutations, most information could be gained from comparing the dhfr haplotypes. However, the risk for treatment failures is not statistically significant when you compare parasites with one, two or three mutations in dhfr as long as they have mutations in dhps. It is notable that most isolates with two dhfr mutations had the combination of S108N + N51I while the S108N + C59R combination was very rare. Even with five mutations (N51I + C59R + S108N for dhfr, and A437G + K540E for dhps) in the parasite folate enzymes, most patients showed ACR. However, the fact that 25% ofcases have parasites that carry the quintuple mutations is amatter of concern for the future use of SP.
Table 2 Clinical and parasitological outcomes n (%) The clinical and parasitological assessments in this study were completed at the end of year 2000, around the time when Uganda’s drug policy changed from CQ mono- therapy to SP + CQ. The aim of the study was to monitor the efficacy of SP, transfer technology for mutation analysis to Makerere University and establish baseline data for mutations in key codons of P. falciparum dhfr and dhps genes. These mutations have been regarded as linked to increased risk for treatment failures due to SP drug pressurebut the dynamics in vivo are not well understood. We notethat while evidence-supported information on anti-mal- showed a more worrying picture (Table 3). A high arial drug efficacy in Uganda has long been limited, frequency of DHFR 108 mutations was not surprising since nationwide compliance with newly introduced policy this has been found repeatedly in many African countries.
changes requires meticulous sensitization of health pro- Frequencies for mutations at DHFR codons 51 and 59 viders throughout the country and effective compliance to were lower but there were a substantial number of mixed the SP + CQ regimen is clearly yet to be assured. Accord- (MX) infections showing both wild type (WT) and mutant ingly, SP or CQ monotherapy is still commonly used in (MT) alleles. More surprising were the very high frequen- Uganda. Yet, only limited information is available on the cies of mutation at DHPS codons 437 and 540, which is prevalence of genetic markers for SP resistance in the even higher than in a recent study at Mulago Hospital in country. A search of previous data only found three Kampala (Dorsey et al. 2003). Considering earlier indica- publications (Jelinek et al. 1999a,b; Dorsey et al. 2003).
tions of triple dhfr mutations plus double dhps mutations Our present study provides important baseline information (the quintuple mutation genotype) as indicators for SP on the prevalence of these markers. Future similar work treatment failures (Kublin et al. 2002), we present the data may then offer useful insights on the rate of development of as haplotypes for the different combinations of mutations resistance to SP in the region. According to our results and have tried to correlate the number of mutations with (Table 2), the efficacy of SP is still satisfactory but morphisms in DHFR and DHPS. Successfulcompleted assays for the three codons Numbers (percentages in brackets) of wildtype (WT), mixed (MX) and mutant (MT) allelesare given.
Tropical Medicine and International Health H. Sendagire et al. Plasmodium falciparum and molecular resistance markers Table 4 Frequencies of the different mutant haplotypes found and show a correlation with treatment failure (Kublin et al.
correlation with clinical response, expressed as number of treat- 2002). We note that from the present study, no single haplotype, on its own, can be implicated in increasing therisk of treatment failure. This may be because of the limited number of treatment failures, but it could also be because of the short follow-up period of 14 days. Our ongoing study where patients were followed-up for 28 days shows more failures, even when you consider that over 30% of these failures are likely to be because of re-infection (Dorsey et al. 2002). We, therefore, recommend that future studies adapt longer follow-up time than 14 days.
Although we have no direct experimental evidence, we speculate that at least some of the treatment failures may be because of host factors including inadequate immune response (Domarle et al. 1999). Our future studies will set up in vitro drug inhibition assays to gauge the role of host factors in treatment failure. Molecular genotyping ofparasite dhfr and dhps genes was not a satisfactory complementary treatment is needed for occasional treat- predictor of clinical and parasitological outcome in this SP ment failures. The presence of dhfr and dhps mutations, study, a result which future surveillance of parasite especially the quintuple mutation, is a sign of warning for resistance to SP in intermittent treatment of pregnancy the future. The most striking result is the very high malaria will need to consider carefully (Shulman et al. 1999).
frequency of mutations in the dhps codons 437 and 540.
Interestingly, recent parasite population studies (Roper Just considering samples with pure mutations, both dhps et al. 2003) have revealed that a unique micro-satellite A437G and K540E were found in higher frequencies than pattern can identify the quintuple mutations which are all dhfr mutations. If samples with mixes of wild type and supposedly associated with treatment failures in Southern mutant are included, dhfr S108N is the most frequent Africa. It may be that the quintuple mutations in them- mutation, with both dhps A437G and K540E still scoring selves do not confer high-level resistance to SP but that the higher than dhfr N51I and C59R. The frequencies of micro-satellite marker is a more useful tool to study linkage DHPS mutations are higher than those reported from between parasite markers and treatment failures. Further another study in Uganda (Dorsey et al. 2003). This might studies are needed to identify the relevant markers.
be due to differences in sulpha-drug pressure on the Continuation of clinical studies for the period 2001– parasite strains investigated in the two separate study 2003 after the SP + CQ combination was adopted as first- populations. Dorsey et al. (2003) studied patients at line treatment showed no dramatic increase in treatment Mulago Hospital in the urban city of Kampala. In contrast, failures (to be published), indicating that high level SP our study was performed at Kasangati Health Clinic, resistant parasites are still not common in Uganda and use which, being peri-urban, largely attracts patients from of SP within combination therapy could be a viable surrounding semi-rural villages where non-prescribed alternative while keeping track of warning signs for medication is less common than in urban areas.
appearance of drug resistance markers. The results from Nevertheless, in both the Mulago and our Kasangati our study and others’ investigations in nine sentinel sites studies, there were more dhps than dhfr C59R mutations.
around Uganda reveal the existence of low to moderate This result disagrees with the earlier proposed hypothesis levels of P. falciparum resistance to SP in different districts (Kublin et al. 2002) that dhfr mutations are selected first of Uganda (Dorsey et al. 2002; Kamya et al. 2002; Roper and dhps mutations appear later. One possible explanation et al. 2003). Added to widespread resistance of P. falcip- for the high frequency of dhps mutations is that previous arm to CQ in the central and east African region (Bloland use of sulphamethoxazole–trimethoprim combinations for et al. 1993; Kamya et al. 2002), the increase in SP bacterial infections may have selected for the dhps muta- resistance is clearly of notable concern for the long term tions found here (Iyer et al. 2001).
use of SP + CQ. Nevertheless, SP is still widely used in Although the treatment failures (Table 2) in this study Uganda either alone as prophylaxis for intermittent treat- are not alarmingly high yet, the presence of 25% of cases ment of malaria in pregnancy or in combination with CQ with the quintuple mutation is of course worrying partic- as policy treatment for uncomplicated malaria. Thus, our ularly because the quintuple mutation has been reported to results provide valuable information on the efficacy of an Tropical Medicine and International Health H. Sendagire et al. Plasmodium falciparum and molecular resistance markers anti-malaria drug which, in practice, is still widely used.
in a longitudinal antimalarial drug efficacy study: comparison of We note that after this work was completed, MOH was in results based on genotyping of msp-1, msp-2, and glurp.
advanced stages of discussions to modify policy such that American Journal of Tropical Medicine and Hygiene 68, 133– while SP will continue to be reserved for intermittent presumptive treatment (IPT) in pregnancy, artemisinin- Domarle O, Migot-Nabias F, Mvoukani JL et al. (1999) Factors influencing resistance to reinfection with Plasmodium falci- based combinations (ACTs) may be recommended to parum. American Journal of Tropical Medicine and Hygiene replace SP + CQ for policy-based cure of uncomplicated malaria in Uganda. Still, looking at the gradual pace at Dorsey G, Njama D, Kamya MR et al. (2002) Sulfadoxine/pyri- which SP + CQ replaced CQ monotherapy in the country methamine alone or with amodiaquine or artesunate for treat- during 2000–2004, it will likely take over 5 years before ment of uncomplicated malaria: a longitudinal randomised trial.
ACTs can be widely available to the population. Accord- ingly, over the coming decade, limited surveillance studies Dorsey G, Vlahos J, Kamya MR, Staedke SG & Rosenthal PJ on the efficacy of SP should be undertaken to guide drug (2003) Prevention of increasing rates of treatment failure by combining sulfadoxine-pyrimethamine with artesunate oramodiaquine for the sequential treatment of malaria. Journal ofInfectious Diseases 188, 1231–1238.
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extra funding provided to the Faculty of Medicine and the Iyer JK, Milhous WK, Cortese JF, Kublin JG & Plowe CV (2001) School of Post Graduate Studies, Makerere University by Plasmodium falciparum cross-resistance between trimethoprim the Swedish Agency for Research Cooperation (SIDA/ and pyrimethamine. Lancet 358, 1066–1067.
SAREC) and for financial support awarded to HS by the Jelinek T, Kilian AH, Curtis J et al. (1999a) Plasmodium falci- Fogarty International Center (FIC). We thank Dr Moses parum: selection of serine 108 of dihydrofolate reductase during Kamya who acts as co-supervisor to HS, and the clinical treatment of uncomplicated malaria with co-trimoxazole in team that participated in the study including Dr C.
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WHO Expert Committee on Malaria (2000) World Health Shulman CE, Dorman EK, Cutts F et al. (1999) Intermittent sul- Organization Technical Report Series, Vol. 892, WHO, Geneva, phadoxine-pyrimethamine to prevent severe anaemia secondary AuthorsHakim Sendagire, Fred Kironde and Daniel Kyabayinze, Department of Biochemistry, Makerere University, Kampala, Uganda.
E-mail: [email protected]; [email protected]; [email protected]¨te Swedberg (corresponding author), Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
E-mail: [email protected]


Department of Mathematics & StatisticsTIME: 3 hours. You are permitted to use one 8 1/2 × 11 “cribsheet”, but no other notesor papers, in this examination. 1. In a study of human blood types in non-human primates, a sample (assume it wasrandom!) of 71 orangutans were tested and 14 were found to have blood type B. (a) Construct a 90% confidence interval for the proportion of in

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