Drug Resistant Malaria Due to Plasmodium Falciparum Clinical Trial
Official title:
Efficacy of Artesunate Monotherapy and Dihydroartemisinin - Piperaquine in Patients With Uncomplicated Falciparum Malaria in Central Vietnam
At the end of 2012, the Institute of Tropical Medicine in collaboration with National
Institute of Malariology Parasitology and Entomology (NIMPE) conducted a study in Quang Nam
province, central Vietnam, to assess the efficacy of the national DHA-PPQ regimen for the
treatment of uncomplicated P. falciparum malaria infections, both in adults and in children.
Results showed that about 30% of the study participants were parasitaemic at day 3. Parasite
clearance rate was estimated at 6.2h, which was comparable to figures from Pailin, Cambodia,
where artemisinin resistance were previously reported . However, results from this study
have to be interpreted bearing in mind that: (i) the age-based drug dosing scheme used has
been criticized as insufficient to clear parasites and (ii) DHA-PPQ drugs used (Artecan™),
Vietnam, are not produced under Good Manufacturing Practices (GMPs). However, those results
prompted the NMCP and WHO to declare Quang Nam, Binh Phuoc, Dak Nong, and Gia Lai provinces
as a "Tier I area" (credible evidence of artemisinin resistance) in May 2013. By end of 2014
a fifth province, Khanh Hoa, was declared Tier I (Dr Hong, Personal Communication). Except
for the south-eastern province of Binh Phuoc, artemisinin resistance has never been
confirmed with an artemisinin based monotherapy in Central Vietnam.
Therefore, in order to confirm artemisinin resistance in Central Vietnam , a study with oral
artemisinin-based monotherapy, using WHO prequalified AS and DHA-PPQ and recommended dosing
scheme of 4mg/kg/day for AS and DHA, is needed. In the arm where study participants are
treated with 3 days of AS monotherapy, treatment will be followed by an additional 3-day
course of DHA-PPQ to effectively clear all parasites.
The aim of the present study is to confirm artemisinin resistance in Central Vietnam by
assessing P. falciparum clearance time and rate after AS monotherapy (WHO recommended
dosage). The investigators will conduct a two-arm open label, randomized study, with one arm
receiving AS monotherapy for 3 days + 3-day of DHA-PPQ, and a second arm receiving 3 days of
DHA-PPQ.
3.1 Study Design This study is designed as a randomized open label 42-day follow-up study to
evaluate the clinical and parasitological responses after treatment of uncomplicated
P.falciparum infections with either AS or DHA-PPQ. Symptomatic patients with P.falciparum
mono-infections, fulfilling the study criteria, will be enrolled into the study, randomized
to one of the treatment arms and followed up for 42 days. All drug administration will be
directly observed and the patients will be actively followed-up for 42 days according to an
extended WHO protocol. At the end of the follow-up time, all patients carrying gametocytes
will be treated with a single dose of Primaquine (0.75mg/kg) following national guidelines.
3.2. Study site and population The study will be carried out in Krong Pa district, Gia Lai
province where both P.falciparum and P.vivax malaria incidences are the highest in the
country. The study will be located in Chu R'Cam commune where the study team will be working
together with the local health staff and all surrounding communes health centers with
malaria patients.
The local population is mainly composed by the Gia Rai ethnic minority whose main
occupations consist of slash and burn agriculture (mainly maize, manioc and rice) in forest
fields, together with seasonal work in rubber plantations, and small-scale production of
goods for daily subsistence or trade e.g. coffee and cashew nuts. The climate is tropical
with an the dry season from November to April and the rainy season from May to October. The
area is hilly and forested (secondary forest) and the main malaria vector is Anopheles
dirus.
3.4. Trial Population The target population includes all P. falciparum infected patients
either presenting spontaneously to the CHC or being referred by the health staff of the
surrounding CHCs.
3.5. Trial procedures 3.5.1 Screening. All consulting patients who meet the basic enrolment
criteria during screening will be assigned a consecutive screening number and evaluated in
greater depth by the medical doctor. Once the patient meets all the enrolment criteria, he
or she (or a parent or guardian in case of children) will be asked for consent to
participate in the study and will be allocated a study number (ID=sequential numbering). Any
person who decides not to participate in the study will be examined, treated and followed-up
by the health facility staff according to the standard of care established by the Ministry
of Health.
3.5.2. Randomisation and Treatment Allocation Randomization will be carried out in blocks of
10 with an allocation ratio of 1:1.
At enrolment, the patient will be interviewed and examined physically and a standardized
pre-coded questionnaire will be filled in. A venous blood sample will be collected into
sterile vacutainer tubes containing EDTA or acid-citrate-dextrose (EDTA/ACD) from all
patients at enrolment. A minimum of 5ml of blood -and maximum of 8 ml- will be collected
from adults, and 1ml/kg up to 5ml will be collected from children. Two blood drops will be
added into a 1.5 ml tube containing 500 µL of RNAprotect reagent (Qiagen).
3.5.3 Treatment All patients will receive a full treatment of either AS alone (4mg/kg/day)
or DHA-PPQ (target dose DHA=4mg/kg/day) for 3 days in order to evaluate early parasite
clearance. Since a 3-day course of AS is insufficient to treat P.falciparum, it will be
completed by an additional 3-day DHA-PPQ regimen according to WHO recommendations. Patients
will be observed for 60 minutes after treatment for adverse reactions or vomiting. Those
patients vomiting their medication within the first 30 minutes will receive a repeat full
dose; those vomiting from 30-60 minutes will receive half dose.
Rescue medications will be given following national guidelines:
Rescue oral drug: Quinine (30mg/kg/day) and clindamycine (15mg/kg/day) for 7days; Rescue IV
drug: Quinine (30mg/kg/day) and doxycycline (3mg/kg/day) for 7days 3.5.4. Follow up From Day
0 until parasite clearance: after the first dose of antimalaria drug has been administered
and observed for one hour, patients will be monitored every 12 hours by blood microscopy at
least until day 3 and longer if necessary until parasite clearance defined as two
consecutive negative slides. At each visit, the patient will be interviewed and examined
physically, the follow-up form completed and any adverse event documented. A finger prick
blood sample will be collected for the following outcomes: two blood slides for LM
observation (thick and thin film), 200 µl of blood into an EDTA/ACD microtainer tube for
later DNA extraction, and 2 drops of blood into another microtainer tube containing
RNAprotect for later RNA extraction. An additional drop of blood will be taken at Day 3 to
measure Hb concentration.
Day 7, 14, 21, 28, 35, 42 Patients will then be asked to attend the clinic (or be visited at
home) for each follow-up visit, during which they will be interviewed and examined, the
follow-up form will be completed, and any adverse event documented. A finger prick blood
sample will be collected for the following outcomes: two blood slides for LM observation
(thick and thin film), 200μl of blood in an EDTA/ACD microtainer tube for DNA extraction,
and 2 drops of blood in RNAprotect containing microtainer tube. At Day 7, 14, 28 and 42, one
drop of blood will be taken for Hb concentration (Hemocue).
Day of P. falciparum recurrence Recurrence is defined as any P.falciparum parasitaemia
detected by LM examination during the patient's follow-up period and after initial parasite
clearance. If recurrence is confirmed a further venous blood sample will be collected
(minimum 5ml/maximum 8ml) prior to rescue treatment administration: ex-vivo assays will be
done with one part of the blood while the remaining will be cryopreserved for future
investigations on drug resistance mechanisms.
3.5.5. Loss to follow-up & protocol violations Loss to follow-up occurs when, despite all
reasonable efforts, an enrolled patient does not attend the scheduled visits and cannot be
found. No treatment outcome will be assigned to these patients. These patients will be
classified as lost to follow-up and censored or excluded from the analysis. Study patients
who meet any of the following criteria will be classified as withdrawn: i) withdrawal of
consent; ii) failure to complete treatment; iii) enrolment violation; iv) voluntary protocol
violation; v) involuntary protocol violation.
3.5.6 Laboratory procedures and evaluation Microscopy: three slides (thin & thick and thin
films) should be obtained at screening. One slide will be stained rapidly (10% Giemsa for
15min) for initial screening. Once the patient is enrolled, the other two slides will be
stained more carefully (3% Giemsa for 45min). Parasite density will be determined by
counting the number of asexual parasites per 200 white blood cells (WBCs) with a hand tally
counter. If more than 500 parasites have been counted before reaching 200 WBCs, the count
will be stopped after completion of the field. Density, will be expressed as the number of
asexual parasites per µl of blood, will be calculated dividing the number of asexual
parasites by the number of WBCs counted and corrected by the estimated WBCs density
(typically 8000 per µl). When the number of asexual parasites is less than 10 per 200 white
blood cells in follow-up smears, counting will be done against at least 500 white blood
cells. A blood slide will be considered negative when examination of 1000 white blood cells
reveals no asexual parasites. Gametocyte density will be computed by counting the number of
gametocytes per 500 WBCs.
Haemoglobin (Hb) concentration will be measured on whole blood collected into micro-cuvette
using Hemocue following manufacturer instructions.
P. falciparum molecular detection: DNA will be extracted from finger-prick blood samples
collected in microtainers. Molecular confirmation of Plasmodium species (P.vivax,
P.falciparum, P.ovale and P.malariae) at Day 0 will be performed by qPCR targeting 18S
ribosomal gene. The same qPCR method will be used to identify and quantify P. falciparum
infections at Day 0 and follow up samples.
P. falciparum genotyping for recrudescence/new infection: genotyping of the recurrent
infections will be done by characterizing MSP1, MSP2 and GLURP, single-copy genes in the
Plasmodium falciparum genome as described elsewhere.
RNA extraction and RT-qPCR for gametocytes: RNA will be extracted from RNAprotect samples
using affinity columns and DNAse digestion of genomic DNA. To detect and quantify P.
falciparum gametocyte stages a reverse transcription qPCR (RT-qPCR) targeting Pfs25 gene
transcripts will be done on Day 0 and in all follow-up samples, following previously
described protocols with modifications.
Ex vivo drug sensitivity assays: venous blood collected in EDTA/ACD tubes (at Day 0 and Day
of recurrence) will be processed within 6 hours after collection. Blood will be centrifuged
and plasma and buffy coat removed and stored at -20ºC. Red blood cells will be washed twice
in RPMI and resuspended in culture medium (RPMI 1640 LPLF liquid medium) to a 50%
hematocrite blood medium mixture (BMM).
Standard ex vivo drug sensitivity assay: the assessment will follow the WHO guidelines
"Field application of in vitro assay for the sensitivity of human malaria parasites to
antimalarial drugs" for the assessment of the response of P. falciparum to artemisinins".
Ex vivo ring stage survival assays (RSA) In addition to standard ex vivo assays, the
performance of ring stage survival assays (RSA) will be explored following a published
protocol.
Molecular markers of artemisinin resistance: extracted DNA will be used to genotype point
mutations in P. falciparum Kelch propeller domain (K13), which have been recently identified
as candidate resistance molecular markers for artemisinin resistance. Briefly, K13 will be
amplified in a nested PCR using primers described in WWARN procedure INV11
[http://www.wwarn.org/toolkit/procedures]. After agarose gel electrophoresis confirmation,
products will be sequenced and analysed to identify polymorphisms. Alternative upcoming
candidates for artemisinin resistance may also be explored 4. QUALITY ASSURANCE Site
monitoring visits will be scheduled on a regular basis. During these visits, information
recorded on the case report forms (CRFs) will be verified against source documents (eg
laboratory records and clinic registers). After the CRFs forms are collated at the end of
the trial, they will be reviewed for completeness and accuracy. The data will be double
entered on site into an electronic database (Epi Info), where specially designed computer
checks are used to identify data entry discrepancies, invalid data ranges and overall
consistency. All discrepancies will be resolved by reference to the original checked data
collection forms.
All blood films on admission will be read at the study site as well as by a senior
microscopist at a coordinating centre. In addition, an external quality control will be done
at ITM on 10% of randomly selected slides. For each of the molecular assays (qPCR, RT-qPCR
and parasite genotyping) 10% of the samples processed at NIMPE will be randomly chosen to be
re-analysed at ITM. External quality control of ex vivo drug assay results will be done at
ITM where the slides will be re-examined for 10% of randomly chosen isolates.
5. Statistical Analysis Plan The parasite clearance time is defined as the elapsed time (in
hours) from the patient's treatment (first dose) time to the first of two consecutive
negative blood slides. Parasite clearance time and rate as well as lag phase will be fitted
using the PCE online tool developed by the WWARN. The analysis of the 42 day cure rate will
be performed both for the modified intention-to-treat (mITT) patient population (all
randomised patients) and the evaluable patient population (Per Protocol). Patients are
evaluable for the analysis of the 28/42 day cure rate if parasite counts are recorded up to
Day 26/40 or the patient discontinues due to "Unsatisfactory therapeutic effect" because of
reappearance of parasites. The mITT analysis also includes patients who discontinue before
Day 28/42 due to reasons other than "Unsatisfactory therapeutic effect" (e.g. this could
include "Adverse experience(s)" because of repeated vomiting). These patients will be
counted in the mITT analysis as treatment failures regardless of their reason for
discontinuation. Patients who had concomitant treatment with antibiotics which have an anti
malarial effect will be excluded from the evaluable patients and included in the mITT.
For categorical variables percentages and corresponding 95% confidence intervals will be
calculated using Wilson's method. Proportions will be compared by calculating the *2 with
Yates' correction or by Fisher's exact test where appropriate. Normally distributed
continuous data will be summarized as means and standard deviations, and compared by
Student's t test and analysis of variance. Data not conforming to a normal distribution will
be summarized as medians (IQR) or geometric means and compared by the Mann-Whitney U test or
Kruskal-Wallis analysis of variance. The association between 2 continuous variables will be
assessed using Spearman's rank correlation coefficient.
The risk of treatment failure by day28 and 42 will all be evaluated by survival analysis
with cumulative incidences calculated by Kaplan Meier method and compared by the
Mantel-Haenszel log rank test.
Parasite, fever and gametocyte clearance times will also be evaluated by Kaplan Meier
method.
Hematological changes during the first week of treatment as well as recovery during weekly
follow-up will be evaluated by measuring the changes in Hb concentration between day0, 3, 7,
and day14, 28 and 42, respectively.
6. ETHICAL ISSUES Ethical clearances Ethical clearance to conduct the study will have been
obtained from the Ethics Committee of the National Institute of Malariology, Parasitology
and Entomology, Hanoi and of the University of Antwerp, as well as from the Institutional
Review Board at the ITM, Antwerp.
Informed consent Patients will be included in the study only if they (or parents or
guardians of children) give informed consent. The consent request is available in English
and translated into Vietnamese language will be read entirely to the patient, parent or
guardian. Details about the trial and its benefits and potential risks will be explained.
Once any questions have been answered, a signature will be requested on the document. If the
patient is illiterate, a literate witness must sign; if possible, the signatory will be
selected by the participant and will have no connection to the research team.
;
Allocation: Randomized, Endpoint Classification: Safety/Efficacy Study, Intervention Model: Parallel Assignment, Masking: Open Label, Primary Purpose: Treatment
| Status | Clinical Trial | Phase | |
|---|---|---|---|
| Completed |
NCT02974348 -
Antimalaria Drugs Susceptibility Testing for an Effective Management of Infected Patients in Sub-Sahara Africa
|
Phase 3 |