Efficacy and safety of artemisinin-based combination therapy, and molecular markers for artemisinin and piperaquine resistance in Mainland Tanzania

Open-label, one-arm prospective studies (N = 8) were conducted to evaluate the clinical and parasitological responses to directly observed treatment of AL and ASAQ in 2011, AL in 2012, and AL and DHAPQ in 2015 using the WHO protocol for the surveillance of the efficacy of anti-malarial drugs [18]. The studies were conducted between March and September, covering the peak transmission season (June–August). They were carried out in the nine National Malaria Control Programme (NMCP) sentinel sites in eight regions; Chamwino in Dodoma, Butimba in Mwanza, Kibaha and Rufiji in Pwani, Kyela in Mbeya, Kilombero in Morogoro, Muheza in Tanga, Nagaga in Mtwara, and Ujiji in Kigoma (Fig. 1). Type of anti-malarial tested per site was agreed upon by all investigators ahead of protocol’s ethics approvals. In 2011 ASAQ was evaluated in Ujiji and Kibaha and AL in Kilombero and Muheza. Due to low number of patients enrolled in 2011, the Tanzania first-line anti-malarial (AL) was recommended to be assessed at all four sites (Butimba, Chamwino, Nagaga and Kyela) in 2012 while in 2015, AL and DHAPQ were evaluated only in Kyela and Rufiji, respectively.

Patients were enrolled if they had mono-infection of P. falciparum detected by microscopy, axillary temperature ≥ 37.5 °C or history of fever during the past 24 h, ability to swallow oral medication, ability and willingness to comply with the study protocol and visit schedule for the duration of the study and informed consent from the parent or guardian of the enrolled children. The age of the patients enrolled varied slightly in the different years: 6–59 months in 2011; 6 months–10 years in 2012 and 6 months and above in 2015. Similarly, the threshold of parasitaemia at enrolment varied from 1000 to 200,000 asexual/µl asexual forms in 2011 and in 2012 to 250–200,000 asexual/µl in 2015. WHO protocol allows to adjust age and enrolment parasitaemia of study patients according to the transmission levels [18]. ACT is contraindicated in the first trimester of pregnancy and, therefore, the WHO protocol requires that all females who reached menarche to be tested for pregnancy. For the study in 2015 female minors aged 12–17 years were excluded from the study as confidentiality and privacy required for pregnancy testing for this age group could not be ensured. Any of the following features was regarded as an exclusion criteria: presence of general danger signs or signs of severe falciparum malaria according to the definitions of WHO, severe malnutrition, febrile conditions due to diseases other than malaria or other known underlying chronic or severe diseases, regular medication, which may interfere with anti-malarial treatment, history of hypersensitivity reactions or contraindications to any of the medicine(s) being tested or used as alternative treatment(s).

At the enrollment, home address, demographic information and full medical history were taken from each patient. Body weight was measured and axillary temperature was taken. Physical examination was done to rule out danger signs and other non-malaria illnesses as per exclusion criteria.

All eligible patients were treated with oral AL, ASAQ or DHAPQ using the WHO recommended therapeutic dose regimens [19]. All treatment doses were given under direct supervision of the study clinician or study nurse in health facilities and participants were observed for 30 min for adverse reactions or vomiting. For AL arm, participants residing far from the health facilities were retained at the facility for the night doses and allowed to go home in the morning. The daily dose was re-administered in case of vomiting within 30 min after drug intake, one child was withdrawn from the study and given the rescue medication after vomiting the replacement dose. Children with fever ≥ 38.5 °C were treated with paracetamol and tepid sponging was also used for those below 5 years. Patients were assessed clinically and parasitology according to the WHO protocol [18] and were followed up for 28 days for treatment with AL and ASAQ, and 42 days for DHAPQ.

Thick and thin smears were taken on days 0 (before treatment) and during subsequent prescribed follow-up on day 1 (if presented with danger signs or severe symptoms), 2, 3, 7, 14, 21, 28 days in 28 days for AL and ASAQ whereas for DHAPQ up to day 42 days. Microscopy examination of thick and thin blood films using Giemsa stain was undertaken to determine parasite species and density according to the WHO protocol [18]. Parasite density (per µl) was calculated assuming a white blood cell count of 8000/µl. All slides were read independently by two qualified microscopists and the average of the two counts was taken as the parasite density. Parasite counts with discordant results (differences in species, presence of parasites or parasite density > 50%) were re-read by a third independent microscopist, and parasite densities were calculated by averaging the two most close counts.

Two to three drops of blood were collected on filter paper (Whatman no. 3 mm, GE Healthcare UK Limited, UK) on day 0 (before treatment) and during recurrence of parasitaemia on day 7 onwards. Each dried blood spots (DBS) was punched with a sterile puncher and placed in a 96 well plate in numerical order. Samples were lysed overnight in a saponin solution, and then, DNAs were extracted with the Instagen Matrix resin as previously described [20]. All DNA samples from patients undergoing symptoms reoccurrence were analysed for genotyping of the highly polymorphic regions msp1, msp2 and glurp loci, as recommended by WHO [21]. DNA samples were also analysed for the presence of mutations in the propeller domain in the k13 gene associated to artemisinin resistance [16]: a portion of the k13 gene was amplified using a nested PCR assay, amplicons (codons 443–666, i.e. 720 bp) were sent to Macrogen Korea for sequencing, and DNA sequences were analysed to identify specific single-nucleotide polymorphism (SNP) related to artemisinin resistance. Samples from the DHAPQ treated patients were also analysed for amplification of plasmepsin2 gene recently associated to piperaquine resistances [17].

Treatment outcomes were determined according to the classification of WHO [18] as early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) and adequate clinical and parasitological response (ACPR). The proportion of patients who were still positive on day 3 was also recorded. Following PCR correction, treatment failure was classified as recrudescence if recurrent parasitaemia was the same parasite strain as of day 0 or new infection if they were different parasite strain. Adverse events including serious adverse events were assessed through physical examination and history taking.

An expected treatment failure rate of 5% was assumed for all the three drugs evaluated and with a 95% confidence level and a precision of 5%, a sample size of 73 was targeted per site or per drug for 2011, 2012 and 2015 studies. An additional 20% was added to ensure that the sample size would be achieved after the exclusion of patients due to loss to follow-up or withdrawal. A total of 88 patients per drug per site were therefore targeted. Data were double entered and analysed using the WHO-designed Microsoft Excel^® program (Microsoft Corporation, Washington). Chi-square test or the Fisher’s exact test (where cell counts were ≤ 5) were used for comparing differences in categorical data. Arithmetic and geometric (only for parasite density) means were considered.

Proportions of treatment failures (ETF, LCF and LPF) and cure rates before and after PCR correction were calculated as per-protocol analysis and Kaplan–Meier analysis. Re-infections and non-interpretable PCR results were excluded from the per-protocol analysis, but retained in the Kaplan–Meier analysis.

These studies were approved by the Ifakara Health Institute (IHI) ethics review committee (ERC), the National Medical Research Coordinating Committee (MRCC) of the National Institute for Medical Research (NIMR) and by the WHO research ethics review committee for studies conducted in 2015.

All English consent forms were translated into Swahili and back translated by a native speaker. Before enrollment, parents or guardians of children below 18 years were asked for a written informed consent. In addition, an assent was sought for all children between 12 and 17 years. For illiterate parents or guardians, a literate witness who was not part of the study team was chosen to sign on his or her behalf. All the information obtained was kept confidential and shared with study team only when it was necessary. Identification numbers were used instead of patients’ names in the case report forms and computer based data entry.