Surveillance of the efficacy of artemisinin-piperaquine for the treatment of uncomplicated Plasmodium falciparum among children under five years in Est Mono district, Togo, 2017

Background Malaria is a major public health problem in and the launched a nationwide artemisinin compound Mass Drug Administration Project in East Mono with a population of 150,000. Before launching the project, the sensitivity test of artemisinin piperaquine tablet was conducted in Elawagnon general clinic. On this background, we evaluated the efficacy and safety of artemisinin piperaquine in the treatment of uncomplicated falciparum malaria in children under five years of age. Methods In this study, children aged 6-59 months without complications of falciparum malaria were observed, and the selected cases were treated with artemisinin piperaquine. The patients were followed up for 28 days to observe the fever clearance time, parasitemia, gametophyte, cure rate, haemoglobin and msp-2 gene polymorphism. The primary end point was the 28-day cure rate, and PCR corrected reinfection and recrudescence. This research was conducted according to standardized WHO protocol for the assessment of the efficacy of anti-malarial treatment. Results A total of 91 children participated in the study. The adequate clinical and parasitological response (ACPR) before PCR-corrected were 66 (72.52%) and 90 (98.90%) after PCR-corrected. The patient was well tolerated to artemisinin piperaquine and no serious adverse reactions were observed. The average hemoglobin level increased by 0.05g/dl per day (p< 0.0001). The gametophyte doesn’t declined at the beginning of treatment, however, 14 days later, it dropped(D21:p<0. 05; D28: p< 0. 01). In the msp-2 gene polymorphism

study of 24 children with positive parasite after treatment, 1 case of msp-2 with 3D7 haplotype and FC27 haplotype was reported, indicating that it's recrudescence, with a frequency of 4.2% (1/24); The others maybe reinfection, with a frequency of 95.8% (23/24). clinical studies showed that it had 97% efficacy in treating uncomplicated falciparum malaria [3][4][5][6]. MHSST decided to conduct sensitivity tests for artemisinin piperaquine at the general clinic in Elawagnon county before launching the universal MDA. Among this background, we evaluated the efficacy and safety of artemisinin piperaquine in the treatment of uncomplicated falciparum malaria in children under five years of age in Elawagnon, Prefecture on Est-Mono province of Togo.

Study sites
Est Mono district covers an area of 2,474 km 2 with a population of 89,060 inhabitants and a population growth rate of 1.03% in 2010 [7]. Est Mono is one of the nine health districts in the Plateaux region, which is one of Togo's six regions.
The district is situated in central Togo and has 17 health facilities run by nurses, auxiliary nurses, or non-qualified nurses. Each health unit has CHWs trained in health promotion and the management of uncomplicated malaria cases within their communities.
Est Mono district [7] and the other central and southern parts of Togo have two rainy seasons, the first starting in April and ending in July and the second starting in

Study design
The evaluation study of artemisinin piperaquine for treatment of uncomplicated falciparum malaria followed the drug sensitivity observation method developed by the World Health Organization(WHO) for 28 days [8]. Based on the studies that have been conducted, the proportion of failed treatments for artemisinin piperaquine is estimated at 5%. To ensure that 95% of the cases were treated successfully and about 5% couldn't participate, at least 73 people were selected for this study as a sample size and conducted at the study site. If 10% of the patients were lost and excluded, the sample size should be 80 persons [8,9]. Therefore, at least 80 people should be included as the target population for the study site, and the maximum number of patients lost should be avoided.

Inclusion criteria
Children 6 to 59 months old, the body temperature greater than or equal to 37.5 °C , no danger signs or symptoms of severe malaria, without obvious symptoms of fever or other symptoms of severe malnutrition (e.g. upper arm length < 11 cm, height and weight ratio < 70%, systemic edema, to uncover her nakedness) were all included, at the same time, with single infection caused by plasmodium falciparum parasite, the population between 2000/μl and 250 000/μl, with the ability of oral drugs, received a parent or legal guardian of informed consent, convenient to clinic review, and with no history or allergic reaction of artemisinin piperaquine contraindications.

Medicine and administration
Artemisinin piperaquine tablets, containing 62.5mg of artemisinin and 375mg of piperaquine each tablet, were administered 1/2 tablet every 24 hours over 2 days for children aged 6 to 24 months; 3/4 tablet every 24 hours over 2 days for children aged 25 to 59 months. The medicines were administered under supervision.
Artemisinin piperaquine was purchased from Artepharm, Co., Ltd (China), with batch number 20160601. All patients in the study were given acetaminophen treatment every 6 hours until the fever symptoms were completely resolved.

Laboratory inspection
Blood samples from finger tips were collected on D0, D2, D3, D7, D14, D21, and D28, respectively, to make thin and thick blood samples. The number of asexual bodies per cubic millimeter of blood was calculated before treatment. No asexual protozoa or gametophyte could be found in 500 fields of thick blood membrane as negative [8]. The amount of hemoglobin was measured on days D0, D7, D14, D21, and D28 using the Hemoglobin Analyzer URIT-12. The filter paper blood pieces were made on D0, D7, D14, D21 and D28, and thin and thick blood glass and filter paper blood pieces were made for laboratory testing in patients who were positive with parasite. At the same time, approximately 3mL of venous blood samples were taken at D0 and D28 of positive patients, using heparin vacuum tubes and stored at -20°C in the laboratory, for white blood cells (WBC) and PCR analysis of polymorphism of merozoite surface protein-2 (msp-2) of plasmodium falciparum to distinguish between recrudescence, and reinfection [10].

Outcomes evaluation
Clinical outcome evaluation was classified according to WHO [8] score:

Early treatment failure (ETF)
Danger signs or severe malaria on D1, D2 or D3, in the presence of parasitaemia; parasitaemia on D2 higher than on D0, irrespective of axillary temperature; parasitaemia on D3 with axillary temperature≥37.5°C; and parasitaemia on D3≥25% of count on D0.

Late clinical failure (LCF)
Danger signs or severe malaria in the presence of parasitaemia on any day between D4 and D28 (day 42) in patients who did not previously meet any of the criteria of early treatment failure; and presence of parasitaemia on any day between D4 and D28 with axillary temperature ≥ 37.5 °C in patients who did not previously meet any of the criteria of early treatment failure.

Late parasitological failure (LPF)
Presence of parasitaemia on any day between D7 and D28 with axillary temperature < 37.5 °C in patients who did not previously meet any of the criteria of early treatment failure or late clinical failure.

Adequate clinical and parasitological response (ACPR)
Absence of parasitaemia on D28, irrespective of axillary temperature, inpatients who did not previously meet any of the criteria of early treatment failure, late clinical failure or late parasitological failure.
Every time after administration, ask whether there are any adverse reactions, such as mental disorder, insomnia, headache, tinnitus, deafness, nausea, vomiting, loss of appetite, abdominal pain, diarrhea, itching, rash and other symptoms, and make detailed records according to the records of adverse reactions.

Statistical analysis
All data was entered twice into the Excel spreadsheet. GraphPad PRISM (GraphPad software, USA) was used for all statistical analyses. The Shapiro-Wilk normality test was used to test the normality of the data. For normally distributed data, t-test was used for comparison between the two groups, while Welch's correction was used at the same time; and One-way ANOVA and Tukey's test were used for multiple comparisons between multiple groups. For non-parametric data, Mann-Whitney test was used for comparison between two groups and Kruskal-Wallis and Dunn test was used for comparison between multiple groups. Bidirectional analysis of variance was used for two -dimensional grouping data.

Study profile and baseline characteristics
Among 208 children, 6 to 59 months of age, with fever screened, 73(35.10%) was negative by microscopy, 7(3.36%) for mixed infections with plasmodium ovale and plasmodium malariae, 33(15.86%) for parasitaemia less than 2000 parasites/ mm 3 , the other 4 (1.92%)children were not included for the following reasons: 2 children for the difficulty in completing the 28-day follow-up because of the long journey; and of the 28-day follow-up, 1 child for receiving other antimalarial treatment on the first day, and 1 child for being failed to follow. And in the end, 91(43.75%) patients were included in this study (Fig. 1).
Baseline clinical and laboratory characteristics of the child subjects were shown in Parasitemia and fever clearance time After AP treatment, most of the patients experienced rapid remission of clinical symptoms ( Fig. 2A), from an average body temperature of 38.5 °C on D0 to 36.5 °C on D2 (p < 0.0001), with 79 cases (87%) below 37 °C. On D2 after administration, 66 (73%) of the patients were observed cleareance of parasites on blood smears, and complete clearance was observed on D3 (Fig. 2B).

Gametophyte and hematological parameters
In the gametophyte observation ( Fig. 3A and 3B) it was found that the average gametocytaemia still increased before the D7, meanwhile the number of patients with gametophyte did not decrease significantly. Both of which decreased synchronously after D7, and significantly down on D21(p < 0.05) and D28(p < 0.01).
While gametophytes were still observed on blood smears on D28 in 6 patients, with a mean value of 8/ul.
The hemoglobin level in the patient's was improved progressively from D0 to D14, D21and D28 (Fig. 3C)   Polymorphism of msp-2 gene and clinical parameters of 24 positive children Two alleles gene (3D7 and FC27) were detected in 24 children who were still positive after taking AP, using nested PCR to analyze the msp-2 gene of all the isolates before and after administration. Only 1 patient, whose 3D7 and FC27 haplotype of msp-2 were the same before and after taking the drug, suggested recrudescence, with a frequency of 4.2% (1/24). Other 23 patients may be reinfection with a frequency of 95.8% (23/24).

Adverse events
In this study, the most common adverse reaction was cough, followed by diarrhea and rash of all the 91 children taking AP. Other notable adverse events included loss of appetite, vomiting and abdominal pain. No serious adverse reactions occurred and AP was well tolerated. The list of adverse events observed during the study was shown in Table 3. There are few studies on the efficacy of malaria treatment and resistance of Artemisinin Combination Therapies (ACTs) in Togo. Only one article on the effect of ACTs was found [3]. The therapeutic effect of AL(artemether -benflumetol) and

DISCUSSION
ASAQ (artesunate -amodiaquine) were monitored in national malaria control  [14]. Overall, parasite clearance reduction, gametophytic carrier rates reduction were consistent with improvements of hemoglobin levels in all 91 patients administrated. After PCR genotyping proofreading, the efficacy threshold of artemisinin piperaquine obtained in our study reached 98.90%, higher than the 95% efficacy threshold recommended by WHO for AL [11]. In addition, artemisinin piperaquine were well tolerated by local children in Togo, and no serious adverse events including diarrhea, weakness, anorexia, hemoglobinuria, itching, or eyelid edema were observed during the 28-day follow-up.
The results are similar to those of the ACTs studies conducted over the past 5 years in Sub-saharan Africa. This combination therapy has a high cure rate for falciparum malaria without complications. A randomized study was [15] conducted in Kenya in 2014, which the PCR-corrected ACPR was 97.8% for AL, and 99.1% for dihydroartemisinin-piperaquine (DP). A single-arm prospective study was [16] conducted in Zambia in 2012, and the results showed that 98% of the patients participating in the study achieved complete clearance of parasitic diseases on D3, and the PCR-corrected ACPR was 100% for AL, that is, all the participating patients achieved ACPR. In a study conducted [17] in three provinces of Angola in 2015, the 28-day cure rate of AL after PCR-corrected was 88.1%-96.3%. No evidence of artemisinin resistance was found in this study. However, pfmdr1 haplotype was found in all AL treatment failures, which was associated with decreased sensitivity of phenylfluorene. A study was [18] conducted in Malawi in 2014, and the cure rate of AL was 69%-82.5% in Non-PCR-corrected, but PCR-corrected APCR was 98%-100%, which was also associated with high reinfection. The authors attributed and performed the PCR analysis. XH, WG, CL, MC, JG, YY and QX collected the data and conducted the research. JS conceived and designed the study, conducted the research. CD conducted the research. All authors read and approved the final manuscript. Temperature and parasitemia across days after treatment. Y axis represents the temperature