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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
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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-
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on the efficacy of SP should be undertaken to guide drug
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The clinical work was carried out with support of WHO/
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TDR re-entry grant no. 981094 to FK. We are grateful for
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extra funding provided to the Faculty of Medicine and the
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School of Post Graduate Studies, Makerere University by
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SAREC) and for financial support awarded to HS by the
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Fogarty International Center (FIC). We thank Dr Moses
parum: selection of serine 108 of dihydrofolate reductase during
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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
PROF. ANDREA LUIGI TRANQUILLI C U R R I C U L U M V I T A E INFORMAZIONI PERSONALI Nome ANDREA LUIGI TRANQUILLI Qualifica Professore Universitario di ruolo (I fascia) SSD MED 40 Ginecologia ed Ostetricia presso l Università Politecnica delle Marche, Ancona Indirizzo Clinica Ostetrica e Ginecologica - Istituto di Scienze Materne ed Infantili Ospedale Salesi Via F