Baseline characteristics of participants enrolled and completing follow-up
A total of 966 participants were screened from July to October 2016 at the two study sites. A total of 211 and 210 participants were included in the Maferinyah and Labé sites, respectively, for a total of 421 participants. Baseline characteristics of the included participants who completed their follow-up are shown in Table 1. At both study sites, nearly all included participants completed their follow-up, with combined exclusion and loss to follow-up rates of less than 3% across all arms. The median age of the study participants was 36 months (interquartile range [IQR]: 24-48) for both study arms in Maferinyah. In Labé, median patient age was higher in the ASAQ arm (45 months, [IQR]: 24-59) than in the AL arm (36 months, [IQR]: 24-48). The median weight of the participants at both sites was similar across all arms, at approximately 13 kg.
Overall, there were fewer girls than boys among the included participants at both sites (39.5% vs 60,5% in Labé and 44.5% vs 55.5% in Maferinyah)
At inclusion (D0), the median baseline parasite density for participants in AL arm was higher at 19,400 parasites/μL ([IQR]: 10,100-51,900) than for those in ASAQ arm 27,430 parasites/μL ([IQR]: 10,215-55,045) in Maferinyah. In Labé, median baseline parasite density was almost the same in both arms at 30,200 and 30,480 parasites/μL respectively. The median haemoglobin level of participants, was 9 g/dl for both arms in Maferinyah 10 g/dl for both arms in Labé.
Table 1. Baseline characteristics of participants enrolled and completing follow-up (n=421)
Variables
|
Maferinyah (n=211)
|
Labé (n=210)
|
ASAQ
|
AL
|
ASAQ
|
AL
|
Participant characteristics at baseline
|
|
|
|
|
Median age, in month (interquartile range)
|
36 (24-48)
|
36 (24-59)
|
45 (24-59)
|
36 (24-48)
|
Median weight, kg (interquartile range)
|
14 (10-16)
|
13 (11-16)
|
13 (10-16)
|
13 (10-15)
|
Percent female, n (%)
|
55 (51.4)
|
39 (37.5)
|
40 (38.1)
|
43 (41.0)
|
Median day 0 parasitemia, parasites/µL*103 (interquartile range)
|
19,4 (101-519)
|
27,4 (10,2-55,0)
|
30,2 (13,0-63,9)
|
30,450(13,1-49,6)
|
Median day 0 haemoglobin, g/ dl (interquartile range)
|
9.7 (8-10)
|
9.8 (8-10)
|
10.7 (9-11)
|
10.6 (8.5-11)
|
AL artemether–lumefantrine, ASAQ artesunate–amodiaquine
Rates of follow up and treatment outcomes of participants who completed their follow-up
Of the 421 included participants, 8 (1.9%) were lost to follow-up, including two deaths. This left 413 participants completing their follow-up. Treatment outcomes of participants who completed their follow-up are shown in Table 2. No ETF was observed in any study arms. In contrast, 22/413 (5.3%) participants developed a late treatment failure: 5/105 (4.8%) in the Maferinyah ASAQ arm, 8/101 (7.9%) in the Maferinyah AL arm, 3/104 (2.8%) in the Labé ASAQ arm and 6/103 (5.8%) in Labé AL arm.
Of the 22 late treatment failures, 2 were classified as recrudescences and 20 were classified as reinfections (Supplemental Table S1). Both recrudescences occurred at D28 of follow-up, one in the Labé AL arm and one in the Labé ASAQ arm. The Kaplan-Meier estimate of the D28 corrected efficacy rate was 100% (CI: 100 - 100%) for the Maferinyah AL arm, 100% (CI: 100 - 100%) for Maferinyah ASAQ arm, 99% (CI: 97.1 - 100%) for the Labé AL arm, and 99% (CI: 97.2 - 100%) for Labé ASAQ arm.
Table 2 Treatment outcomes for participants finishing follow-up as part of therapeutic efficacy monitoring in Guinea, 2016 (N=413)
|
Maferinyah
|
Labé
|
|
ASAQ
|
AL
|
ASAQ
|
AL
|
Enrolled
|
107
|
104
|
105
|
105
|
Reached study outcome
|
105
|
101
|
104
|
103
|
Treatment failure
|
5 (5)
|
8 (8)
|
3 (3)
|
6 (6)
|
Early treatment failure
|
0
|
0
|
0
|
0
|
Late treatment failure
|
5 (5)
|
8 (8)
|
3 (3)
|
6 (6)
|
Recrudescence
|
0
|
0
|
1 (1)
|
1 (1)
|
Day 28
|
0
|
0
|
1 (1)
|
1 (1)
|
Reinfection
|
5 (5)
|
8 (8)
|
2 (2)
|
5 (5)
|
Day 21
|
1 (1)
|
3 (3)
|
1 (1)
|
1 (1)
|
Day 28
|
4 (4)
|
5 (5)
|
1 (1)
|
4 (4)
|
Adequate clinical and parasitological response
|
100 (95)
|
93 (92)
|
101 (97)
|
97 (94)
|
Kaplan-Meier Day 28 efficacy
|
|
|
|
|
Uncorrected
|
95.2% (91.3-99.4)
|
92.1% (87.1-97.5)
|
97.1% (94-100)
|
94.2% (89.9-98.8)
|
Microsatellite-corrected
|
100% *
|
100%*
|
99% (97.2-100)
|
99% (97.1-100)
|
AL artemether–lumefantrine, ASAQ artesunate–amodiaquine, 28-day follow-up
*confidence intervals: undefined
Safety of the anti-malarial drugs and deaths observed
Vomiting at any time during treatment was observed in 59/421 (14.0%) participants with 26 (44.1%) in the AL arm and 33 (53.9%) in the ASAQ arm. Two participants (one in the Maferinyah ASAQ arm and one in the Maferinyah AL arm) developed signs of severe malaria less than 24 hours after inclusion in the study; one (in the Maferinyah AL arm) ultimately died. Both were excluded from analysis due to onset of severe symptoms less than 24 hours after inclusion following WHO definitions. One additional participant in the Maferinyah ASAQ arm died from a car accident during follow-up, and was censored at day 14 in the analysis.
Results of Day 2 and Day 3 microscopy
The proportion of negative slides at D2 and D3 of follow-up is shown in Table 3. At D2 of follow-up, the vast majority of the participants had negative slides at examination at both sites and arms (>88% for the two arms in Maferinyah and > 90% in Labé). At D3 of follow-up, nearly all slides were negative at both sites and in both arms (>99% in Maferinyah and 100% in Labé).
Table 3 Proportion of slides negative for asexual malaria parasites on days 2 and 3 following treatment (n=413)
Variables
|
Maferinyah (n=206)
|
Labé (n=207)
|
ASAQ
|
%
|
AL
|
%
|
ASAQ
|
%
|
AL
|
%
|
Day 2
|
94
|
89.5
|
89
|
88.1
|
99
|
95.2
|
94
|
91.3
|
Day 3
|
103
|
99.0
|
102
|
100
|
103
|
100
|
100
|
100
|
Molecular markers of resistance
DNA was isolated from 443 samples, including 421 pretreatment and 22 day-of-late treatment failure samples. Of the 443 samples analysed, 411 (93%) were successfully amplified and sequenced for pfk13 (Table 4). The majority of D0 (98%, 380/389) and all day-of-late treatment failure (100%, 22/22) samples were wild type for pfk13. All 9 observed pfk13 mutations were polymorphisms that have not been associated with artemisinin resistance.
Amplification and sequencing of the pfmdr1 gene at the 86, 184, and 1246 codons was successful in 380/443 samples (86%). The NFD haplotype was the predominant pfmdr1 haplotype at D0, present in 54% (197/362) of analysable D0 samples, followed by the NYD haplotype, present in 44% (158/362) of D0 samples. In late treatment failure samples, the NFD haplotype also predominated, at 61% (11/18).
Table 4 Prevalence of molecular markers of resistance in Day 0 and Day of Failure samples from therapeutic efficacy studies in Guinea, 2016
|
|
Day 0
|
Day of Failure
|
pfk13
|
n=389
|
n = 22
|
Wild-type
|
380 (98%)
|
22 (100%)
|
Mutant
|
9 (1%)*
|
0 (0%)
|
pfmdr1 86 codon
|
n=379
|
n =18
|
N
|
296 (78%)
|
14 (78%)
|
Y
|
59 (16%)
|
4 (22%)
|
N/Y
|
24 (6%)
|
0 (0%)
|
pfmdr1 184 codon
|
n=379
|
n=18
|
Y
|
117 (31%)
|
3 (17%)
|
F
|
206 (54%)
|
14 (78%)
|
Y/F
|
56 (15%)
|
1 (6%)
|
pfmdr1 1246 codon
|
n=370
|
n=18
|
D
|
363 (98%)
|
18 (100%)
|
Y
|
6 (2%)
|
0 (0%)
|
D/Y
|
1 (0.3%)
|
0 (0%)
|
pfmdr1 haplotypes**
|
n=362
|
n=18
|
NYD
|
158 (44%)
|
4 (22%)
|
YYD
|
17 (5%)
|
0 (0%)
|
NFD
|
197 (54%)
|
11 (61%)
|
YFD
|
71 (20%)
|
4 (22%)
|
NYY
|
1 (0.3%)
|
0 (0%)
|
YYY
|
6 (2%)
|
0 (0%)
|
NFY
|
1 (0.3%)
|
0 (0%)
|
YFY
|
0 (0%)
|
0 (0%)
|
*All 9 samples with polymorphisms not associated with artemisinin resistance: P419S (1), L429L (1), C469C (3), G496G (1), E509E (1), V510V (1), A621A (1)
**Haplotypes defined at codons 86, 184, and 1246. Samples with multiple haplotypes were included in the numerator for each haplotype