Factors contributing to delay in parasite clearance in uncomplicated falciparum malaria in children.

Abstract

Drug resistance in Plasmodium falciparum is common in many endemic and other settings but there is no clear recommendation on when to change therapy when there is delay in parasite clearance after initiation of therapy in African children.
The factors contributing to delay in parasite clearance, defined as a clearance time > 2 d, in falciparum malaria were characterized in 2,752 prospectively studied children treated with anti-malarial drugs between 1996 and 2008.
1,237 of 2,752 children (45%) had delay in parasite clearance. Overall 211 children (17%) with delay in clearance subsequently failed therapy and they constituted 72% of those who had drug failure, i.e., 211 of 291 children. The following were independent risk factors for delay in parasite clearance at enrolment: age less than or equal to 2 years (Adjusted odds ratio [AOR] = 2.13, 95% confidence interval [CI]1.44-3.15, P < 0.0001), presence of fever (AOR = 1.33, 95% CI = 1.04-1.69, P = 0.019), parasitaemia >50,000/ul (AOR = 2.21, 95% CI = 1.77-2.75, P < 0.0001), and enrolment before year 2000 (AOR= 1.55, 95% CI = 1.22-1.96, P < 0.0001). Following treatment, a body temperature >or= 38 degrees C and parasitaemia > 20000/microl a day after treatment began, were independent risk factors for delay in clearance. Non-artemisinin monotherapies were associated with delay in clearance and treatment failures, and in those treated with chloroquine or amodiaquine, with pfmdr 1/pfcrt mutants. Delay in clearance significantly increased gametocyte carriage (P < 0.0001).
Delay in parasite clearance is multifactorial, is related to drug resistance and treatment failure in uncomplicated malaria and has implications for malaria control efforts in sub-Saharan Africa.

Full text

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RESEARCHOpen Access
Factors contributing to delay in parasite
clearance in uncomplicated falciparum
malaria in children
Akintunde Sowunmi1,2*, Elsie O Adewoye3, Grace O Gbotsho1,2, Christian T Happi2, Abayomi Sijuade1,
Onikepe A Folarin2, Titilope M Okuboyejo1, Obaro S Michael1,2
Abstract
Background: Drug resistance in Plasmodium falciparum is common in many endemic and other settings but there
is no clear recommendation on when to change therapy when there is delay in parasite clearance after initiation
of therapy in African children.
Methods: The factors contributing to delay in parasite clearance, defined as a clearance time > 2 d, in falciparum
malaria were characterized in 2,752 prospectively studied children treated with anti-malarial drugs between 1996
and 2008.
Results: 1,237 of 2,752 children (45%) had delay in parasite clearance. Overall 211 children (17%) with delay in
clearance subsequently failed therapy and they constituted 72% of those who had drug failure, i.e., 211 of 291
children. The following were independent risk factors for delay in parasite clearance at enrolment: age less than or
equal to 2 years (Adjusted odds ratio [AOR] = 2.13, 95% confidence interval [CI]1.44-3.15, P < 0.0001), presence of
fever (AOR = 1.33, 95% CI = 1.04-1.69, P = 0.019), parasitaemia >50,000/ul (AOR = 2.21, 95% CI = 1.77-2.75,
P < 0.0001), and enrolment before year 2000 (AOR= 1.55, 95% CI = 1.22-1.96, P < 0.0001). Following treatment,
a body temperature ≥ 38°C and parasitaemia > 20000/μl a day after treatment began, were independent risk
factors for delay in clearance. Non-artemisinin monotherapies were associated with delay in clearance and
treatment failures, and in those treated with chloroquine or amodiaquine, with pfmdr 1/pfcrt mutants. Delay in
clearance significantly increased gametocyte carriage (P < 0.0001).
Conclusion: Delay in parasite clearance is multifactorial, is related to drug resistance and treatment failure in
uncomplicated malaria and has implications for malaria control efforts in sub-Saharan Africa.
Background
The emergence and spread of multidrug resistance in
Plasmodium falciparum is a major obstacle to successful
chemotherapeutic control of the disease. Resistance to
chloroquine (CQ) and sulphadoxine-pyrimethamine (SP)
is now widespread in sub-Saharan Africa, South Asia
and South America [1,2] and there is an increased
chance that resistance to mefloquine (MQ), already
widespread in Southeast Asia [3-6], may spread to
Africa. With increasing failure of amodiaquine (AQ) in
areas of intense transmission [7-9] and increasing
selection of P. falciparum multidrug resistance gene 1
(Pfmdr 1) in asexual and sexual parasites following treat-
ment of infections with artemether-lumefantrine (AL)
[10], there is a rising spectre of reduced responses to
artemisinin-based combination therapy (ACT) in Africa.
Despite increasing drug treatment failure, there is no
clear guidelines, at least in Nigeria, about the time to
change anti-malarial drug treatment if parasites do not
clear quickly from peripheral blood following treatment
of uncomplicated acute infections in African children. It
is postulated in the present study that, parasite clearance
exceeding two days is associated with risk of treatment
failure and resistance and could be used as a criterion
to change therapy in very young children. The present
* Correspondence: akinsowunmi@hotmail.com
1Department of Pharmacology & Therapeutics, University of Ibadan, Ibadan,
Nigeria
Sowunmi et al. Malaria Journal 2010, 9:53
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study reports the relationship between delay in parasite
clearance and anti-malarial treatment failure in
children with falciparum malaria in an area of intense
transmission in south-western Nigeria, where resistance
in P. falciparum to CQ and SP deteriorated steadily
over the past ten years.
Methods
Patients
The studies took place between July 1996 and July 2008
in patients presenting at the University College Hospital
in Ibadan, a hyperendemic area for malaria in south-
western Nigeria [11]. Ethical clearance was provided by
the local ethics committee. During the period, a series
of anti-malarial drug studies were conducted to evaluate
the efficacy and safety of different treatment regimens
(Table 1). Details of the drug efficacy studies have been
described before [12-16].
Briefly, children with symptoms compatible with acute
falciparum malaria who fulfilled the following criteria
were enlisted in the study: age 144 months or below,
mono-infection with P. falciparum, parasitaemia ≥ 2,000
asexual forms/μl blood, negative urine tests for anti-
malarial drugs 4-aminoquinolines and sulphonamides
(Dill-Glazko and lignin tests, respectively), absence of
concomitant illness, no evidence of severe malaria [17],
and written informed consent given by parents or guar-
dians. After enrolment and start of treatment (day 0),
follow-up with clinical and parasitological evaluation
was at days 1-7, and then on days 14 - 28 up to 2004.
After 2004, follow-up was for 42 d. Clinical evaluation
consisted of a general clinical examination including
measurement of weight, core temperature and physical
examination.
Assessment of parasitaemia
Thick and thin blood films prepared from a finger prick
were Giemsa-stained and were examined by light micro-
scopy under an oil-immersion objective, at × 1,000 mag-
nification, by two independent assessors. Parasitaemia in
thick films was estimated by counting asexual parasites
relative to 1,000 leukocytes, or 500 asexual forms,
whichever occurred first. From this figure, the parasite
density was calculated assuming a leukocyte count of
6,000/μl of blood. Gametocytes were also counted in
thick blood films against 1,000 leukocytes assuming an
average leukocyte count of 6,000/μl of blood [18-20].
Haematocrit was done at enrolment in 994 of the
Table 1 Treatment regimens and time of study in the children enrolled
Drugs*Regimens†
No of patientsYear
AQ
AQAS
AQPS
30 mg/kg of amodiaquine base over 3 days, that is, 10 mg/kg daily
Artesunate given as 4 mg/kg dail for 3 d plus amodiaquine given as in AQ above
Amodiaquine given as in AQ above plus sulphadoxine-pyrimethamine given as 25 mg/kg
of the sulphadoxine component at presentation
Amodiaquine given as in AQ above plus sulphalene-pyrimethamine given as 25 mg/kg
of the sulphalene component at presentation
Mefloquine given as 25 mg/kg at presentation plus artesunate as given in AQAS above
Artemether (20 mg) plus lumefantrine (120 mg) given thus: 5-14 kg received 1 tab.,
15-24 kg received 2 tab., 25-34 kg received 3 tab., > 34 kg received4 tab.
at presentation, 8 h later and at 24, 36, 48 and 60 h after first dose
Artesunate given as 28 mg/kg over 7 days, that is, 4 mg/kg daily
Co-trimoxazole given as 20 mg of the sulphamethoxazole component twice daily
30 mg/kg of chloroquine base over 3 days, that is, 10 mg/kg daily
30 mg/kg of chloroquine base over 3 days, that is, 10 mg/kg daily plus chlorpheniramine 8 mg
start and 4 mg 8 hourly for 5 d.
30 mg/kg of CQ base over 3 days, i.e., 10 mg/kg daily plus ketotifen 25 mg/kg statim,
followed by 0.125 mg/kg 8 hourly for 4 d.
30 mg/kg of amodiaquine base over 3 days, that is, 10 mg/kg daily plus sulphadoxine-pyrimethamine
given as 25 mg/kg of the sulphadoxine component at presentation
Mefloquine given as 25 mg/kg at presentation
Pyrimethamine-sulphadoxine given as 25 mg/kg of the sulphadoxine component at presentation
Sulphadoxine-pyrimethamine given as in PS above plus probenecid at 20-25 mg/kg in two divided
doses daily for 3 day
573
142
69
2000-2006
2004-2005
2000
AQSP 91 2006
AMQ
AL
174
90
2007-2008
2006
AS
COT
CQ
CQCP
120
104
388
315
2006
1998-1999
1996-2004
1996-1999
CQKET702001
CQPS1072000/2004
MQ
SP
SPP
176
291
42
2007-2008
1996-2004
2004
† All drugs were administered orally. AQ, amodiaquine; AQART, amodiaquine plus artesunate; AQPS, amodiaquine plus pyrimethamine-sulphadoxine; AQSP,
amodiaquine-sulphalene-pyrimethamine; ARTMF, mefloquine plus artesunate; ARTMLUM, artemether plus lumefantrine; AS, artesunate; COT, cotrimoxazole; CQ,
chloroquine; CQCP, chloroquine plus chlorpheniramine; CQKET, chloroquine plus ketotifine; CQPS, chloroquine plus pyrimethamine-sulphadoxine; MQ,
mefloquine; PS, pyrimethamine-sulphadoxine; PSP, pyrimethamine-sulphadoxine plus probenecid;
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patients treated with non-artemisinin combination ther-
apy (NACT) or ACT in order to evaluate the safety of
combination anti-malarial therapies.
Evaluation of response to drug treatment
Response to drug treatment was assessed using World
Health Organization (WHO) criteria [21] as follows: S =
sensitive, clearance of parasitaemia without recurrence;
RI (mild resistance) = parasitaemia disappears but reap-
pears within 7 to 14 days; RII (moderate resistance) =
decrease of parasitaemia but no complete clearance
from peripheral blood; RIII (severe resistance) = no pro-
nounced decrease or increase in parasitaemia at 48
hours after treatment. In those with sensitive or RI
response, parasite clearance time (PCT) was defined as
the time elapsing from drug administration until there
was no patent parasitaemia for at least 72 h. Delay in
parasite clearance was defined as a parasite clearance
time > 2 d, and was based on the asexual life cycle of 48
h in the infected erythrocyte [22]. The above criteria
used for assessing responses to anti-malarials were used
in order to maintain uniformity and because they were
the recommended criteria when studies started.
Molecular analysis of pfmdr 1 and pfcrt
At enrolment, 100 μl of capillary blood was collected on
to 3 MM Whatman™ filter paper for resistance markers
determination in 2000 and 2006 in those treated with
CQ or AQ. Pfmdr 1 and pfcrt genotypes were assessed
by PCR using methods described previously [7,10]. Sin-
gle nucleotide polymorphisms in pfmdr 1 and pfcrt were
detected by nested PCR-RFLP methods.
Statistical analysis
Data were analysed using version 6 of the Epi-Info soft-
ware [23], and the statistical program SPSS for Windows
version 10.01 [24]. Proportions were compared by calcu-
lating c2with Yates’ correction. Normally distributed,
continuous data were compared by Student’s t-tests and
analysis of variance (ANOVA). Data not conforming to
a normal distribution were compared by the Mann-
Whitney U-test and the Kruskal-Wallis test (or by Wil-
coxon rank sum test). A multiple logistic regression
model was used to test the association between parasite
clearance > 2 d (yes or no at presentation or during fol-
low up) and factors that were significant at univariate
analysis: age, presence of fever, asexual parasitaemia at
presentation or during follow-up, a history of vomiting,
and drug treatment. Because the study was conducted
over a period of 12 years, time in years since the com-
mencement of trials was included as a covariate in the
model for pretreatment delay in parasite clearance. P-
values of < 0.05 were taken to indicate significant
differences.
Results
Between July 1996 and July 2008, 2,752 children (1342
females) were enrolled into the drug studies. All were
recruited into prospective randomized studies and all had
primary infections with P. falciparum. There were 1,716
under five-year olds. The geometric mean parasitaemia
at enrolment was 34,044/μl (95% CI 30,400 - 33,664)
(Table 2). Follow up was achieved in 2,562 children for
up to 21 days and in 2,122 children for 28 or 42 days.
Treatment failures
Overall 57 of the 2,752 children (2.1%) had treatment
failure by day 7, and this rose to 113/2,695 (4.2%) by
day 14, and 62/796 (7.8%) by day 21 (c2for trend =
155.8, P < 0.0001).
Drug treatment and delay in parasite clearance
Overall, delay in parasite clearance occurred in 1237 of
the 2752 children (45%) (Figure 1). The highest propor-
tions of children showing delay in parasite clearance
were found in those treated with CQ (70.8%), PS
(63.9%), CQCP (60%), COT (58.6%) AQ (52%) CQKET
(51.4%) CQPS (42%), AQPS (37.6%), AQSP (37.3%), PSP
(30.9%), and MQ (22.7%). The proportions of children
with delay in parasite clearance were significantly lower
in those treated with AS (2.5%), AQAS (9.9%), AL
(6.6%) and, AMQ (5.7%) when compared with the latter
group above (c2= 447.91, P < 0.0001). There was no
significant difference in the proportions of children with
delay in clearance in those treated with AS (3 of 120),
AQAS (14 of 142), AL (6 of 90), and AMQ (10 of 174)
Table 2 Baseline clinical and parasitological parameters
of the 2752 children enrolled in the study
VariablesMean ± SD
(range)
95% CI
Age (year)
No. < 5 years
Weight (kg)
Axillary temperature (°C) (n = 2428) 38.3 ± 1.2 (34-42)
No. with > 40°C
Haematocrit (%) (n = 994)
No. with < 30%
Parasitaemia (/μl)
GM
Range
No with > 100,000 (/μl)
No. with >250,000 (/μl)
Gametocytaemia (/μl) GM
Duration of illness (d)
Duration of vomiting (d)
6.1 ± 3.0 (0.5-12)
1716 (63%)
17.3 ± 6.4 (5-47)
6.0-6.2
17.0-17.6
38.2-38.3
210
30.5 ± 4.8 (10-51)
380
30.1-30.7
34,044
2009-1,194,285
638 (23.2%)
187 (6.8%)
27 (6 - 4188)
3.0 ± 1.4 (1-14)
1.3 ± 1.4 (1-9)
30,400-33,664
2.9-3.0
1.2-1.3
GM, geometric mean
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(c2= 6.1, df = 3, P = 0.10). The addition of AS to MQ
(AMQ) significantly reduced the proportion of patients
with delay in clearance (40 of 176 for MQ v 10 of 174
for AMQ, c2= 19.24, P = 0.0001). Similarly, the addi-
tion of AS to AQ (AQAS) significantly reduced the pro-
portion of patients with delay in clearance (298 of 573
for AQ v 14 of 142 for AQAS, c2= 80.49, P < 0.0001).
Temporal changes in parasite clearance time and
proportion of children with delay in clearance in those
treated with chloroquine, sulphadoxine-pyrimethamine
and amodiaquine
The changes in parasite clearance times (PCT) and the
proportions of children with delay in clearance between
1996 and 2004, or between 2000 and 2006, in patients
treated with CQ (n = 316), SP (n = 218), or AQ (n = 573)
were evaluated. There was a significant increase in PCT
from 3.08 ± 1.06 d (n = 53) in 1996 to 4.23 ± 2.08 d (n =
76) in 2004 (P < 0.0001), but there was no difference in
proportion with delay in parasite clearance during the
same period (71.7% in 1996 and 80.2% in 2004, c2for
trend = 1.36, P = 0.24) in those treated with CQ (Figure 2).
There was a significant increase in PCT from 3.19 ±
1.72 d (n = 48) in 1996 to 4.21 ± 1.84 d (n = 71) in
2004 (P = 0.0001) and a significant increase in propor-
tion with delay in parasite clearance during the same
period (58.3% in 1996 and 80.3% in 2004, c2for trend =
5.99, P = 0.014) in those treated with SP (Figure 2).
Figure 1 Numbers of children with delay in parasite clearance (> 2 d) following treatment with antimalarial drugs. Total enrolled (Green
line), No. with parasite clearance > 2 d (Red line), No. with parasite clearance > 2 d who proceded to failure (Blue line). [see Table 1 for
antimalarial drug abbreviations].
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There was also a significant increase in PCT from 2.58
± 0.80 d (n = 104) in 2000 to 2.87 ± 1.39 d (n = 118) in
2006 (P = 0.0001), but there was no difference in pro-
portion with delay in parasite clearance during the same
period (53% in 2000 and 59% in 2006, c2for trend =
0.81, P = 0.36) in those treated with AQ (Figure 2).
Risk factors for delay in parasite clearance at enrolment
The following were found to be independent risk factors
for delay in parasite clearance at enrolment (Table 3):
age ≤ 2 years (Adjusted odds ratio [AOR] = 2.13, 95%
confidence interval [CI] = 1.44-3.15, P < 0.0001), pre-
sence of fever (AOR = 1.33, 95% CI = 1.04-1.69, P =
0.019), parasitaemia >50,000/μl (AOR = 2.21, 95% CI =
1.77-2.75, P < 0.0001), and enrolment before year 2000
(AOR = 1.55, 95% CI = 1.22-1.96, P < 0.0001). A history
of vomiting was associated with an increased risk of
delay in clearance (crude odds ratio = 1.34, 95% CI =
1.07 -1.58, P = 0.009).
Risk factors for delay in parasite clearance following
initiation of treatment
Following treatment, a body temperature ≥ 38°C and
parasitaemia > 20,000/μl blood a day after treatment
began, were independent risk factors for delay in clear-
ance (Table 4). Non-artemisinin monotherapy was asso-
ciated with delay in clearance.
Delay in parasite clearance and treatment failure
Of the 20, 146, 10, 15, 19, and 62 children who failed
treatment with AQ, CQ, COT, CQCP, MQ, and PS,
respectively, 13, 119, 7, 14, 5, and 50 children, respec-
tively were those who previously had delay in parasite
clearance (Figure 3). Overall, 291 children failed treat-
ment. Of these, 211 (72%) had delay in parasite clear-
ance. The latter represent 17% of the total children
(1237) with delay in parasite clearance. Thus delay in
clearance may be related to subsequent drug treatment
failure.
1996 19982000 2002 200420062008
2
3
4
5
40
60
80
100
Year
Mean parasite clearance time (d)
Proportion with PC > 2 d
Figure 2 Temporal changes in parasite clearance time (PCT) and proportion with delay in parasite clearance (PC > 2 d) in children
treated with amodiaquine (AQ), chloroquine (CQ) or pyrimethamine sulphadoxine (PS) between 1996 and 2006. [Proportion of children
treated with AQ who had PC > 2 d (Black line), Proportion of children treated with CQ who had PC > 2 d (X), Proportion of children treated
with PS who had PC > 2 d (O), PCT for AQ treated children (–•–), PCT for CQ treated children (Blue line), PCT for PS treated children (+)]. Bars
represent standard deviation.
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