P. Falciparum Resistance to Artemisinin in Vietnam

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.