Efficacy and Safety of Triple Combination Therapy With Artesunate-Amodiaquine–Methylene Blue for Falciparum Malaria in Children: A Randomized Controlled Trial in Burkina Faso
Efficacy and Safety of Triple Combination Therapy With Artesunate-Amodiaquine- Methylene Blue for Falciparum Malaria in Children: A Randomized Controlled Trial in Burkina Faso
accepted 0 2
electronically published 0
B. C. 0
M. P. contributed equally to this work. 0
0 Ruprecht-Karls-University Heidelberg , INF 324, 69120 Heidelberg, Germany (olaf. The Journal of Infectious Diseases
1 Department of Medical Microbiology, Radboud University Medical Center , Nijmegen , the Netherlands
2 Institute of Tropical Medicine and International Health , Charité-Universitätsmedizin Berlin , Germany
3 Department of Immunology & Infection, London School of Tropical Medicine and Hygiene , United Kingdom
4 Division of Infectious Diseases and Tropical Medicine, Medical Center
5 Centre de Recherche et de la Formation au Paludisme , Ouagadougou , Burkina Faso
6 Centre de Recherche en Santé de Nouna , Nouna
7 Institute of Pharmaceutics and Biopharmaceutics, Heinrich Heine University Düsseldorf
8 Institute of Medical Biometry and Informatics
9 Department of Pediatrics and Adolescent Medicine, Medical School, Ulm University
10 German Center for Infection Research , Partner Site Munich
11 Department of Bacteriology, Max von Pettenkofer-Institute, Ludwig Maximilian University of Munich
12 Biochemistry Centre, Ruprecht-Karls-University , Heidelberg
13 Institute of Public Health, Medical School
Background. Methylene blue (MB) has been shown to be safe and effective against falciparum malaria in Africa and to have pronounced gametocytocidal properties. Methods. Three days of treatment with artesunate (AS)-amodiaquine (AQ) combined with MB was compared with AS-AQ treatment in a randomized controlled phase IIb study; the study included 221 children aged 6-59 months with uncomplicated falciparum malaria in Burkina Faso. The primary end point was gametocyte prevalence during follow-up, as determined by microscopy and real-time quantitative nucleic acid sequence-based amplification (QT-NASBA). Results. The gametocyte prevalence of Plasmodium falciparum at baseline was 3.6% (microscopy) and 97% (QT-NASBA). It was significantly lower in the AS-AQ-MB than in the AS-AQ group on day 7 of follow-up (microscopy, 1.2% vs 8.9% [P < .05]; QT-NASBA, 36.7% vs 63.3% [P < .001]). Hemoglobin values were significantly lower in the AS-AQ-MB group than in the AS-AQ group at days 2 and 7 of follow-up. Vomiting of the study medication occurred significantly more frequently in the AS-AQ-MB group. Conclusions. The combination of MB with an artemisinin-based combination therapy has been confirmed to be effective against the gametocytes of P. falciparum. MB-based combinations need to be compared with primaquinebased combinations, preferably using MB in an improved pediatric formulation. Clinical Trials Registration. NCT01407887.
Combination therapy is the current cornerstone of
malaria control, with the particular aim to delay and
possibly reverse the development of drug resistance
]. Artemisinin-based combination therapy (ACT) is
highly effective, but artemisinin resistance may be
selected in vivo by uncontrolled use of artemisinin
monotherapies or may result from combination with
ineffective partner drugs [
]. The artemisinins also
show pronounced activity against immature Plasmodium
falciparum gametocytes, but they leave mature gametocytes
unaffected and therefore do not prevent malaria transmission shortly
after treatment [
]. There are also differences in
gametocytocidal activity between the available ACT regimens [
Methylene blue (MB) is a water-soluble dye that has been used
for a long time in industry and medicine [
]. It was historically
used to successfully treat malaria [
], but only observational
data supported its efficacy particularly in quinine-refractory
]. In humans, MB is well absorbed from the
gastrointestinal tract, peak plasma concentrations are reached 2 hours after
oral administration, and the plasma half-life is about 20 hours
]. Renal excretion is the major elimination pathway [
Known adverse effects of MB include bitter taste, green or blue
coloration of urine, and mild and self-limiting dysuria [
]. Moreover, a transient reduction of hemoglobin values in
glucose-6-phosphate dehydrogenase (G6PD)–deficient patients but
without overt clinical consequences has been observed in clinical
trials with some 1000 patients in Burkina Faso .
The interest in MB as an antimalarial drug was reactivated
when P. falciparum glutathione reductase was identified as a
new drug target [
]. In a series of studies conducted in Burkina
Faso, oral MB (4–24 mg/kg/d) was shown to be safe and
effective in the treatment of uncomplicated falciparum malaria when
combined with other antimalarials [
] but to act more
slowly against asexual parasites than artemisinins .
Nevertheless, MB has shown a strong effect in terms of P. falciparum
gametocyte reduction [
], matching pharmacokinetics and
potential synergy with artemisinin drugs [
], and a low potential
for resistance development [
]. Consequently, MB has been
considered a potentially useful partner drug for existing ACTs,
particularly in regions where malaria elimination is the final
]. We therefore tested the gametocytocidal effect of
MB when combined with a common ACT,
amodiaquineartesunate (AS-AQ), as well as the efficacy and safety of this
triple combination therapy in children with uncomplicated
falciparum malaria in Burkina Faso.
MATERIALS AND METHODS
The study was conducted from August to October 2011 in the
urban research zone of the Centre de Recherche en Santé de
Nouna (CRSN) in Burkina Faso [
Study Design and Objectives
The study was designed as a randomized controlled phase IIb
trial with follow-up for 28 days. The study was open label but
with blinding for the laboratory technicians and laboratory
scientists involved. The primary objective was to assess the
reduction of P. falciparum gametocytes among children with
uncomplicated malaria after treatment with the triple combination
AS-AQ-MB compared with AS-AQ. The secondary objectives
were to study the safety and the efficacy against asexual parasites
of the 2 regimens. The primary end point of the study was P.
falciparum gametocyte prevalence during follow-up, as assessed by
microscopy and real-time quantitative nucleic acid
sequencebased amplification (QT-NASBA). The secondary end points
were P. falciparum gametocyte density during follow-up as
assessed by microscopy and QT-NASBA, fever and parasite
clearance times, hemoglobin concentrations during follow-up, and
rates of adequate clinical and parasitological response (ACPR),
early treatment failure (ETF), late treatment failure (LCF), and
late parasitological failure (LPF) (both crude and corrected with
polymerase chain reaction [PCR] corrected for reinfections) [
as well as the incidence of observed and self-reported adverse
events (including hemolysis, defined as any drop in hemoglobin
of >2.5 g/dL within 24 hours) and the acceptance of the different
treatment regimens by mothers/caretakers, as determined by a
standardized questionnaire on day 14. Study participants with
ETF, LCF, or late parasitological failure received rescue treatment
with artemether-lumefantrine (Coartem).
The community was informed of the project and invited to
attend central locations in the town of Nouna for fever
measurement. Children who were eligible for the study (see inclusion
criteria) were referred to the Nouna district hospital outpatient
clinic for further examinations. Noneligible sick children
received free treatment in the hospital. The inclusion criteria
were age 6–59 months, weight ≥6 kg, uncomplicated
falciparum malaria (axillary temperature ≥37.5°C or a history of
fever during the last 24 hours and asexual parasitemia [parasite
count, ≥2000/µL and ≤200 000/µL blood]), Burkinabé
nationality, permanent residence in the study area, and informed
consent of parents/caregivers. Exclusion criteria were signs of severe
], moderately severe anemia (hemoglobin, <7 g/dL),
any apparent other disease, and treatment with malaria drugs
during the preceding week.
Study children were randomly assigned to receive either
AS-AQ-MB or AS-AQ during a 3-day period. AS-AQ is the
official first-line antimalarial used in Burkina Faso. Children in
the AS-AQ-MB group received a once-daily tablet of
fixeddosed AS-AQ (Coarsucam; Sanofi Aventis), with dosage
determined by weight group (6.0–8.9 kg, 25 mg of AS plus 67.5 mg of
AQ; 9.0–17.9 kg, 50 mg of AS plus 135 mg of AQ; >17.9 kg, 100
mg AS plus 270 mg of AQ), combined with once-daily MB (15
mg/kg) minitablets in prepackaged sachets according to weight
group (6.0–8.9 kg, 100 mg MB; 9.0–12.9 kg, 150 mg MB; 13.0–
16.9 kg, 200 mg MB; >16.9 kg, 250 mg MB).
Coarsucam was procured from the quality-controlled stock of
the Essential Drug Store, Ministry of Health, Burkina Faso. The
MB minitablets were developed at the Düsseldorf University in
Germany and produced by the Pharbil Waltrop company in
Germany under good manufacturing practice conditions. The
formulation is based on the recently introduced principle of
orodispersible minitablets, which dissolve rapidly in the oral
cavity, preventing aspiration [
]. The blue biconvex minitablets
are 2 mm in diameter and approximately 2 mm thick. They
contain 2.0 mg MB as the active pharmaceutical ingredient,
mannitol/polyvinylacetate (Ludiflash; BASF) as a ready-to-use filler
and binder, sucralose (Merck) as a sweetener, and magnesium
stearate (Peter Greven) as a lubricant.
Coarsucam tablets were taken with water. The MB
minitablets were provided on a spoon with local food to improve the
acceptability for children. All study drugs were administered
under direct observation by study nurses. In case of vomiting
within the first 30 minutes, the treatment was repeated. If
vomiting occurred again, the patient was excluded, referred to the
pediatrics department of the hospital, and replaced. Children
with a temperature ≥38.5°C received a standard dose of
paracetamol (acetaminophen) tablets (10 mg/kg) every 6 hours until
the fever subsided.
Children were recruited on day 0 and followed up on days 1, 2,
3, 7, 14, and 28. Parents and caregivers were advised to come
back at any time between scheduled visits in case of unforeseen
A finger-prick blood sample was taken on days 0, 1, 2, 3, 7, 14,
and 28, and during unscheduled visits. Malaria parasitemia and
Male sex, No. (%)
Age, median (range), mo
Weight, median (range), kg
Duration of current disease episode, median (range), d
Hemoglobin, median (range), g/dL
Temperature, mean (SD), °C
P. falciparum asexual parasites/µL
P. falciparum gametocytemia prevalence, No. (%)
P. falciparum gametocyte density by microscopy,
mean (SD), parasites/µL
P. falciparum gametocyte density by QT-NASBA, median TTP−1
hemoglobin values were determined using standard CRSN
]. In brief, thick and thin blood films were examined
by 2 experienced laboratory technicians supervised by one of
the investigators (B. C.). Asexual parasites and gametocytes
were counted on thick blood films against 200 white blood
cells, and parasite density was calculated assuming an average
white blood cell count of 10 000/mL. Slides were declared
negative if no parasites were seen in 400 fields on the thick film.
Hemoglobin concentrations were measured using a HemoCue
From each blood sample, 2 aliquots (50 µL each) were kept as
dried blood spots on filter paper (Whatman 903; Whatman
International). They were stored in the laboratory of the CRSN
individually sealed in plastic bags with a desiccant. After
shipment to the Centre National de Recherche et de Formation sur
le Paludisme in Ouagadougou, recrudescence was differentiated
from new infection by comparing PCR-generated P. falciparum
merozoite surface protein 1 (msp-1) and 2 (msp-2) genotype
patterns in matched pairs of isolates obtained on admission
and on the day of reappearance of parasitemia, as
recommended by the World Health Organization [
] (see also
Supplementary Material Methods). The interpretation of the results was
based on the consensus definitions of recrudescence and new
At the Ludwig Maximilian University of Munich,
QTNASBA of Pfs25 messenger RNA was performed, as described
in detail elsewhere [
] (see also Supplementary Material
Methods). Owing to a transport-related loss of all filter papers taken
for QT-NASBA analysis, this analysis could only be performed
on 109 backup day 0 and day 7 samples (49 AS-AQ-MB and 60
Calculations were performed with SAS (version 9.1, SAS
Institute Inc., Cary, NC) and Sigma-Stat (version 3.5, Systat
Software, San José, CA) statistical software. Two-sided P values
are reported throughout. For a detailed description of sample
size calculation and statistical tests used, see also the
The study protocol was approved by the ethics committees of
Heidelberg University and the CRSN. Parents/caregivers were
asked for their written consent after having received detailed
information from a study nurse about all known risks and benefits
of the study.
A total of 1029 children were referred from the fever
measurement points to the hospital for assessment, and of those 221
(111 AS-AQ-MB, 110 AS-AQ) were included in the study
(see also Supplementary Figure 1). Seven children refused to
take the first treatment dose (3 AS-AQ-MB, 4 AS-AQ), 18
repeatedly vomited the first treatment dose (15 AS-AQ-MB, 3
AS-AQ), and 3 were erroneously included (1 AS-AQ-MB, 2
AS-AQ). The remaining children (92 AS-AQ-MB, 101 AS-AQ)
formed the a priori defined full analysis set for the modified
intention-to-treat (mITT) analysis. Three cases of minor protocol
violations at enrollment were kept in the per protocol (PP)
analysis: 1 child was 63 months old, 1 had a P. falciparum parasite
count of 1600/µL, and 1 had a hemoglobin value of 6.2 g/dL.
During follow-up, another 10 children were withdrawn from
the study, all from the AS-AQ-MB group. The reasons were
repeated vomiting after the second treatment dose (7 children),
refusal of the second treatment dose (2 children), and protocol
violation (1 child). The remaining children (82 AS-AQ-MB, 101
AS-AQ) form the a priori defined analysis set for the PP analysis.
The demographic and clinical characteristics of the mITT
participants were similar in the 2 study groups (Table 1). The
enrollment prevalence of P. falciparum gametocytes was 3.6%
by microscopy (AS-AQ-MB, 6.5%; AS-AQ, 1.0%) and 97.2%
by QT-NASBA (AS-AQ-MB, 100.0%; AS-AQ, 95.0%).
Microscopic gametocyte prevalence (P = .04) and density (P = .03)
were significantly higher, whereas the gametocyte prevalence
as determined by QT-NASBA was nonsignificantly higher
(P = .25) in the AS-AQ-MB group than in the AS-AQ group
The prevalence of microscopically assessed gametocyte
substantially increased in both study groups from day 0 to day 1 and
continued to do so in the AS-AQ group until day 3. In contrast,
gametocyte prevalence decreased continuously in the
AS-AQMB group from day 1 onward (Figure 1A; Table 2). By day 7,
only 1 patient (1%) in the AS-AQ-MB group had
microscopically detectable gametocytes, and none (0%) by day 14 (AS-AQ,
9% and 5% respectively; P < .05 each). Microscopic gametocyte
densities in gametocyte-positive individuals did not differ
between the 2 treatment groups at baseline or at follow-up dates
(data not shown).
QT-NASBA results were available only for a limited number
of randomly chosen study participants in the AS-AQ (60 paired
day 0 and day 7 samples) and AS-AQ-MB (49 samples) arms.
The available samples indicated that the effect of AS-AQ-MB
on QT-NASBA–detected gametocyte prevalence was even
more pronounced (Table 2). On day 7, gametocyte prevalence
had decreased from 100.0% (49 of 49 samples) to 36.7% (18 of
49 samples) in the AS-AQ-MB arm compared with 97.2% (57
of 60 samples) to 63.3% (38 of 60 samples) in the AS-AQ arm
(P < .001). The reciprocal time to positivity (TTP−1) was
determined for QT-NASBA–positive samples of both groups to
allow for relative density comparison between the groups. The
TTP−1 (indicating gametocyte concentration, calculated only
for QT-NASBA–positive samples) decreased from day 0 to
day 7 in both the AS-AQ-MB (P < .001) and the AS-AQ
(P = .003) arms. On day 7, the median TTP−1 was significantly
lower in the AS-AQ-MB than in the AS-AQ arm (P < .001)
(Figure 1B; Table 2).
Asexual Parasites and Fever Clearance
The clearance of P. falciparum asexual parasites was
nonsignificantly more rapid (1.82 vs 1.96 days; P = .22) in the AS-AQ-MB
than in the AS-AQ group (Table 3). The microscopically
determined parasite prevalence and densities were consistently lower
during day 1 to day 3 in the AS-AQ-MB group than in the
ASAQ group; the difference in parasite density was significant on
day 1 (P = .004). Fever disappeared rapidly in both groups, with
only 6 febrile patients on day 1 (2 AS-AQ-MB, 4 AS-AQ) and 1
on day 2 (AS-AQ-MB).
Prevalence, No. (%)
Prevalence, No. (%)
Prevalence, No. (%)
Prevalence, No. (%)
(N = 92)
The uncorrected rate of ACPR in mITT analysis was 44% in the
AS-AQ-MB and 55% in the AS-AQ group (P = .10). This
difference was largely due to an increased rate of ETF (11%; danger
sign: persistent vomiting) in the AS-AQ-MB group (Table 4).
After correction for reinfections, the rate of ACPR was
significantly lower in the AS-AQ-MB group than in the AS-AQ
group (71.4% vs 85.1%; P = .024); again, this difference was
due to the ETFs in the former group. In the PP analysis,
ACPR occurred at a similar rate in the 2 study groups (80.2%
vs 85.1%; P = .395).
Hemoglobin values decreased from baseline to day 1 in both
groups and steadily increased thereafter in the AS-AQ group
(Figure 2). Values were significantly lower in the AS-AQ-MB
than in the AS-AQ group on day 2 (P = .04) and day 7
(P = .005). There were a total of 6 hemolysis cases (defined as
an hemoglobin drop of >2.5 g/dL within 24 hours), with 3
cases in each treatment group. Five hemolysis cases occurred
between days 0 and 1, and 1 (AS-AQ group) between days 2
and 3. Hemoglobin values rapidly increased thereafter without
Abbreviations: ACPR, adequate clinical and parasitological response; AS-AQ,
artesunate-amodiaquine; AS-AQ-MB, AS-AQ combined with methylene blue;
ETF, early treatment failure; ITT, intention-to-treat; LCF, late treatment failure;
LPF, late parasitological failure.
a This group included 92 patients without correction and 82 with correction.
There were no deaths and only 1 serious adverse event during
the follow-up period. The latter occurred in a 59-month- old
child (AS-AQ-MB) who developed malaria and typhoid fever
on day 17 and recovered after appropriate treatment. The event
was considered unrelated to the study medication.
There were no differences in the number and pattern of
adverse events between the 2 study groups (Table 5). However, it
has to be considered that 25 children vomited the first and
second doses of the study medication and were either excluded
from the study or judged as having ETF. Of these, significantly
more were from the AS-AQ-MB group than from the AS-AQ
group (22 of 92 vs 3 of 101; P < .001).
Despite these findings, no differences in the pattern of
selfreported acceptance of the study medication between groups
were observed. Most mothers considered the treatment to be
good (AS-AQ-MB, 67%; AS-AQ, 65%) or acceptable
(AS-AQMB, 33%; AS-AQ, 35%).
The present data extend our previous findings on the
pronounced gametocytocidal effects of MB [
] by using it in
combination with an ACT and using a molecular gametocyte
detection tool to determine the effect of MB on submicroscopic
gametocyte densities. Posttreatment gametocyte prevalence
was significantly lower in patients treated with
AS-AQMB than in those treated with AS-AQ. When molecular
QT-NASBA gametocyte detection as used in a subset of samples,
AS-AQ-MB–treated children had a 2-fold lower gametocyte
prevalence and density compared with their AS-AQ–treated
Delayed clearance of parasitemia after ACT treatment as
observed in Southeast Asia may favor the transmission of
artemisinin-resistant malaria [
]. Of note, studies in Kenya suggested
a reduction in ACT efficacy in recent years [
] and an
association between parasite clearance time and transmissibility to
]. Against this background, the gametocytocidal
effects of antimalarials are likely to be of increasing importance
in the near future. Primaquine (PQ) is a gametocytocidal
compound with well-known activity against mature P. falciparum
AEs During 28-Day Follow-up by Study Group (ITT
gametocytes but has been associated with intravascular
]. The present study shows that MB may be a viable
alternative to PQ. Similar to the situation with PQ, the infectivity
of gametocytes that persist after MB treatment is currently
unclear and needs further investigation [
One of the main limitations of a number of malaria drugs is
the risk of hemolysis in G6PD-deficient patients [
importance of such effects was recently demonstrated by the
failure of the large chlorproguanil-dapsone (Lapdap) drug
development project [
]. In the present study, the hematological
recovery was less rapid in children treated with AS-AQ-MB
than in those treated with AS-AQ, but there were no differences
in the number of hemolytic events. This supports the notion of
an existing but possibly clinically not relevant hematological
]. Clinical trials directly comparing MB and PQ are
required to finally assess the potential superiority of MB
concerning the risk of hemolysis. Addition of a single low dose of PQ
(0.25 mg base per kilogram) to ACT as a P. falciparum
gametocytocide without requirement for G6PD testing has currently
been recommended by the World Health Organization
for elimination areas or areas threatened by artemisinin
It has been shown that MB acts synergistically with
artemisinins against the asexual blood stage parasites of P. falciparum
]. This in vitro finding was supported by consistent
observations of more rapid parasite clearance during clinical trials in
Burkina Faso [
]. Notably, however, the overall efficacy of
AS-AQ, with or without MB, was comparatively low at 80%–
85% in PP analysis. Previous studies among children with
uncomplicated malaria in the study area yielded PCR-corrected
ACPR rates of 82% for AS-AQ in 2006  and 61% for AQ
alone in 2005 [
]. Impaired AQ efficacy in this area of intense
chloroquine resistance [
] may thus partially explain the
suboptimal efficacy of the ACT. Our findings of a more rapid
clearance of asexual parasites in the AS-AQ-MB group than in the
AS-AQ group may therefore need to be interpreted with some
caution. Future studies should investigate whether the addition
of MB to more efficacious ACTs, such as
artemether-lumefantrine or dihydroartemisinin-piperaquine, also increases the
parasite clearance rates. A more rapid parasite clearance would
probably correspond to a more benign clinical course and a
quicker reduction in the source of any new gametocytes,
which further supports the idea of adding MB to ACTs [
QT-NASBA revealed much higher gametocyte prevalence
than microscopy, which supports similar findings from other
African studies wherein gametocyte prevalence in patients ranged
from 9% to 25% by microscopy but from 68% to 89% by
]. Because even low gametocyte densities can
result in mosquito infection, molecular methods are more
informative than microscopy for determining posttreatment
transmission potential, although both assays fall short of permitting
definitive statements on the transmission-blocking potential of
antimalarial drugs that requires mosquito feeding assays [
The finding that some drugs, including MB, may have differential
activity against male and female gametocytes [
highlights the value of confirming the infectivity of gametocytes that
persist after treatment. Mosquito feeding assays are available only
at a limited number of African research centers.
Although we did not perform feeding assays and had limited
QT-NASBA gametocyte observations owing to an unforeseen
loss of FP samples, we consider our conclusions on the
enhanced gametocyte clearance of the triple combination therapy
justified, given that these are based on both microscopic and
QT-NASA data and that the MB combination was superior at
all time points beyond day 3 after initiation of treatment. The
relatively low efficacy of the AS-AQ combination against
asexual parasites may have influenced gametocyte dynamics during
], although our last time point of gametocyte
detection by QT-NASBA (day 7) is likely to reflect the effect of
MB on gametocyte clearance rather than preventing de novo
gametocyte development, which may take 10–15 days from
persisting asexual parasites [
Oral MB is known to have a strong bitter taste, which may
affect compliance, particularly in the treatment of children.
Compared with aqueous MB solutions, MB minitablets are much
more acceptable for oral intake together with semisolid or liquid
food, but a slightly bitter taste from released MB remains. This is
shown by the fact that in this study significantly more children of
the MB group than in the ACT control group repeatedly vomited
the medication and had to be excluded from the trial or were
judged as having ETF. It is very likely that the observed vomiting
is linked to MB taste attributes only and not to the ingestion of
solid drug carriers as it has been demonstrated for children in the
targeted age group [
]. The World Health Organization
recommended this “multiparticulate” dosage form for the use in hot
and humid climates to improve drug stability and acceptability
and also to reduce transport and storage costs .
In conclusion, MB combined with an ACT could function as
a useful combination for the treatment of falciparum malaria in
elimination programs. Before such a triple therapy is
implemented, the safety and efficacy of this new MB formulation
needs to be compared with other gametocytocidal drugs in
head-to-head clinical trials, and the dosage may need to be
optimized. Minitablets are a promising new pediatric formulation
for MB but may need to be coated for further taste masking to
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org). Supplementary materials consist of
data provided by the author that are published to benefit the reader. The
posted materials are not copyedited. The contents of all supplementary
data are the sole responsibility of the authors. Questions or messages
regarding errors should be addressed to the author.
Acknowledgments. We thank Provepharm Company (Marseille) for the
special provision conditions concerning the MB raw material and David
Poluda for helping with the QT-NASBA analysis.
Author contributions. P. E. M., J. B., R. H. S., F. P. M., C. D., T. B., and
O. M. designed the study. B. C., M. P., N. B. R., A. W., and S. B. S. were
responsible for the laboratory work. M. B., P. E. M., E. N., and A. S. were
responsible for the clinical work. C. K. and M. K. were responsible for data
management and analysis. J. B. developed the MB mini tablets and
organized the production. O. M. wrote the first draft of the paper. All authors
contributed to the interpretation of the data and to writing the manuscript.
Disclaimer. Sponsors had no influence on the study.
Financial support. This work was supported by the German Science
Foundation (Sonderforschungsbereich 544, project A8) and were supported by
the Bill and Melinda Gates Foundation (grant OPP1034789 to C. D. and T. B.).
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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