AAC Accepted Manuscript Posted Online 1 June 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00921-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Lack of artemisinin resistance in Plasmodium falciparum in Uganda based on
2
parasitological and molecular assays
3 4
Roland A. Cooper1*, Melissa D. Conrad2, Quentin D. Watson3, Stephanie J. Huezo1, Harriet
5
Ninsiima4, Patrick Tumwebaze4, Samuel L. Nsobya4, and Philip J. Rosenthal2
6 7
1Department
of Natural Sciences and Mathematics, Dominican University of California, San
8
Rafael, CA, USA
9
2Department
of Medicine, University of California, San Francisco, CA, USA
10
3Department
of Integrative Biology, University of California, Berkeley, CA, USA
11
4Infectious
Diseases Research Collaboration, Kampala, Uganda
12 13
Running Title – Lack of artemisinin resistance in Uganda
14 15 16
*Correspondence – Roland A. Cooper, Department of Natural Sciences and Mathematics,
17
Dominican University of California, 50 Acacia Ave, San Rafael, CA 94901, USA
18
([email protected]).
19 20
1
21
Abstract
22
We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in
23
Kampala in 2014. By standard in vitro assays all isolates were highly sensitive to
24
dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6 hour DHA pulse
25
parasitemia was undetectable in 40/43 cultures at 72 hours. Two of 53 isolates had non-
26
synonymous K13-propeller polymorphisms, but not the mutations associated with
27
resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda.
28 29
Key words – Plasmodium falciparum, malaria, artemisinin-based combination therapy,
30
K13, pfcrt, pfmdr1, drug resistance
2
31
Artemisinin-based combination therapy is now the standard to treat falciparum
32
malaria. However, this regimen is threatened by resistance to artemisinins, manifest as
33
delayed clearance of parasitemia after therapy, in southeast Asia (1, 2). Recently, resistance
34
has been associated with increased parasitemias in culture, compared to those in sensitive
35
parasites, 72 hours after a 6 hour pulse with dihydroartemisinin (DHA), and with
36
polymorphisms in propeller domains of the Plasmodium falciparum kelch (K13;
37
PF3D7_1343700) gene (3-6). Although artemisinin resistance is evident to date only in
38
southeast Asia, the bulk of falciparum malaria occurs in sub-Saharan Africa. Clinical
39
resistance has not been noted in Africa, as rapid parasite clearance has been the rule in
40
many trials, including those in Uganda (7-9). In recent surveys in Uganda (10), and other
41
locations in Africa (11-15), K13-propeller polymorphisms were identified, but these were
42
not the mutations clearly associated with artemisinin resistance in Asian isolates. Results
43
for a new correlate of artemisinin resistance, the ex vivo ring-stage survival assay (3), have
44
not been reported previously for P. falciparum from Africa. We characterized artemisinin
45
sensitivity by this assay and assessed K13 polymorphisms in isolates from Uganda.
46
Parasites were collected from patients diagnosed with malaria from May-August, 2014 at
47
Mulago Hospital, Kampala. Samples were delinked from patient information, so clinical
48
data were unavailable. After malaria diagnosis, excess blood collected in a heparinized tube
49
as part of clinical care was promptly delivered to the laboratory. Giemsa-stained thin
50
smears were made, and samples containing only P. falciparum with parasitemias ≥0.1%
51
were placed in culture after washing of erythrocytes, as previously described (16); blood
52
was also spotted onto filter paper. Samples with >1% parasitemia were diluted with
53
uninfected erythrocytes for a final parasitemia of 1%.
3
54 55
The susceptibility of fresh isolates to DHA and chloroquine was assessed by a
56
standard ex vivo HRP-2 based ELISA microplate assay, as previously described (17), except
57
that drugs were freshly diluted from frozen stocks prior to each assay. Results from single
58
assays were fitted to a variable-slope sigmoidal function using GraphPad Prism 6.0f to
59
generate IC50 values. All data were visually assessed for parasite growth above background
60
and integrity of curve fits.
61 62
Parasite susceptibility to DHA was also assessed using the new ex vivo ring-stage
63
survival assay (3). 20 μl erythrocyte pellets were added to 1.0 ml RPMI 1640 medium
64
supplemented with 25 mM HEPES, 0.2% NaHCO3, 0.1 mM hypoxanthine, 100 μg/ml
65
gentamicin, and 0.5% Albumax I (Invitrogen) containing 700 nM DHA or, for controls, 0.1%
66
DMSO at a hematocrit of 2%. Cultures were incubated at 37°C under 3% O2, 5% CO2, and
67
92% N2, and after 6 hours washed three times with 10 ml RPMI pre-warmed to 37°C, and
68
then placed in drug-free culture media. 72 hours after assay initiation, Giemsa-stained thin
69
smears were prepared. Cultures were considered viable and appropriate for assessment if
70
control parasitemia increased since culture initiation. Parasitemias in control cultures were
71
determined by counting parasites per 1000-2000 erythrocytes. In DHA-pulsed cultures,
72
10,000-20,000 cells were scanned for parasites. RSA survival rates were expressed as the
73
proportion of parasites in the DHA-treated cultures relative to controls, at the end of the 72
74
hour assay. For some samples, cultures were maintained for up to 4 weeks after initiation
75
of the experiment, with smears assessed on alternating days. For positive cultures, blood
76
spots were collected on filter paper.
4
77 78
DNA was extracted from filter paper blood spots using Chelex-100, and gene
79
fragments spanning loci of interest were amplified by nested PCR (10, 18). K13 propeller-
80
encoding domains (codons 440-726) were dideoxy sequenced as previously described (4).
81
To detect polymorphisms in pfcrt and pfmdr1, multiplex ligase detection reaction–
82
fluorescent microsphere assays were performed as previously described (19, 20).
83
Nucleotide sequence data are available in the GenBank database under accession numbers
84
KR055739-KR055804.
85 86
We collected 53 isolates from patients with malaria in May-July 2014. Giemsa-
87
stained thin smears from all blood samples demonstrated only P. falciparum at
88
parasitemias of 0.1-24.3%. We determined ex vivo IC50 data for 15 isolates. Values ranged
89
from 0.4-2.7 nM (geometric mean = 1.6 nM) for DHA and 9.8-1060 nM for chloroquine
90
(Table 1).
91 92
As standard drug sensitivity assays have not identified artemisinin resistance in
93
Asian isolates, we also assessed DHA susceptibility using the ex vivo ring-stage survival
94
assay (3). 10 isolates were lost to contamination or failed to grow. For the remaining 43
95
isolates, assessed 72 hours after culture initiation, most DHA-pulsed cultures contained
96
pyknotic forms, and only 3 of 43 demonstrated healthy-appearing parasites, all with
97
parasitemias ≤0.025%. Of the 3 positive cultures, a single parasite was detected per
98
~15,000 erythrocytes in 2 cultures, and 3 parasites were detected in the third (RSA
99
survival rates of 0.7 - 1.9%). All 43 untreated control cultures demonstrated increased
5
100
parasitemia and healthy-appearing parasites over the course of the assay. Because of the
101
challenges of quantifying extremely low parasitemias, we assessed the viability of 14 DHA-
102
pulsed cultures that did not demonstrate parasites after 72 hours. These cultures were
103
maintained for up to 30 days after initiation of the experiment. 12 of these cultures
104
demonstrated parasites, beginning 15-29 days after culture initiation; two cultures
105
remained negative at 30 days. Thus, we did not observe enhanced growth of DHA-pulsed
106
parasites after 72 hours in culture, arguing against the presence of the artemisinin-
107
resistant phenotype, although most cultures survived the DHA pulse.
108 109
Sequencing the K13-propeller domains from the 53 isolates revealed two
110
nonsynonymous mutations, S552C (mixed with the wild type allele) and A578S, in
111
propeller domains 2 and 4, respectively (Figure 1). Ten isolates that grew in culture 15-29
112
days after the DHA pulse all had wild type K13 sequences. Two of these samples were from
113
isolates that were positive by microscopy 72 hours after the DHA pulse. Considering the
114
putative drug transporters encoded by pfcrt and pfmdr1, polymorphism prevalences were
115
similar to those described recently from other regions of Uganda, with increased
116
prevalence of wild type sequences at pfcrt K76, pfmdr1 N86, and pfmdr1 D1246 compared
117
to older studies (Figure 1) (20, 21). Sensitivity to chloroquine correlated well with the pfcrt
118
K76T sequence, with IC50 values >120 nM for all mutant parasites,
1
Lack of artemisinin resistance in Plasmodium falciparum in Uganda based on
2
parasitological and molecular assays
3 4
Roland A. Cooper1*, Melissa D. Conrad2, Quentin D. Watson3, Stephanie J. Huezo1, Harriet
5
Ninsiima4, Patrick Tumwebaze4, Samuel L. Nsobya4, and Philip J. Rosenthal2
6 7
1Department
of Natural Sciences and Mathematics, Dominican University of California, San
8
Rafael, CA, USA
9
2Department
of Medicine, University of California, San Francisco, CA, USA
10
3Department
of Integrative Biology, University of California, Berkeley, CA, USA
11
4Infectious
Diseases Research Collaboration, Kampala, Uganda
12 13
Running Title – Lack of artemisinin resistance in Uganda
14 15 16
*Correspondence – Roland A. Cooper, Department of Natural Sciences and Mathematics,
17
Dominican University of California, 50 Acacia Ave, San Rafael, CA 94901, USA
18
([email protected]).
19 20
1
21
Abstract
22
We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in
23
Kampala in 2014. By standard in vitro assays all isolates were highly sensitive to
24
dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6 hour DHA pulse
25
parasitemia was undetectable in 40/43 cultures at 72 hours. Two of 53 isolates had non-
26
synonymous K13-propeller polymorphisms, but not the mutations associated with
27
resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda.
28 29
Key words – Plasmodium falciparum, malaria, artemisinin-based combination therapy,
30
K13, pfcrt, pfmdr1, drug resistance
2
31
Artemisinin-based combination therapy is now the standard to treat falciparum
32
malaria. However, this regimen is threatened by resistance to artemisinins, manifest as
33
delayed clearance of parasitemia after therapy, in southeast Asia (1, 2). Recently, resistance
34
has been associated with increased parasitemias in culture, compared to those in sensitive
35
parasites, 72 hours after a 6 hour pulse with dihydroartemisinin (DHA), and with
36
polymorphisms in propeller domains of the Plasmodium falciparum kelch (K13;
37
PF3D7_1343700) gene (3-6). Although artemisinin resistance is evident to date only in
38
southeast Asia, the bulk of falciparum malaria occurs in sub-Saharan Africa. Clinical
39
resistance has not been noted in Africa, as rapid parasite clearance has been the rule in
40
many trials, including those in Uganda (7-9). In recent surveys in Uganda (10), and other
41
locations in Africa (11-15), K13-propeller polymorphisms were identified, but these were
42
not the mutations clearly associated with artemisinin resistance in Asian isolates. Results
43
for a new correlate of artemisinin resistance, the ex vivo ring-stage survival assay (3), have
44
not been reported previously for P. falciparum from Africa. We characterized artemisinin
45
sensitivity by this assay and assessed K13 polymorphisms in isolates from Uganda.
46
Parasites were collected from patients diagnosed with malaria from May-August, 2014 at
47
Mulago Hospital, Kampala. Samples were delinked from patient information, so clinical
48
data were unavailable. After malaria diagnosis, excess blood collected in a heparinized tube
49
as part of clinical care was promptly delivered to the laboratory. Giemsa-stained thin
50
smears were made, and samples containing only P. falciparum with parasitemias ≥0.1%
51
were placed in culture after washing of erythrocytes, as previously described (16); blood
52
was also spotted onto filter paper. Samples with >1% parasitemia were diluted with
53
uninfected erythrocytes for a final parasitemia of 1%.
3
54 55
The susceptibility of fresh isolates to DHA and chloroquine was assessed by a
56
standard ex vivo HRP-2 based ELISA microplate assay, as previously described (17), except
57
that drugs were freshly diluted from frozen stocks prior to each assay. Results from single
58
assays were fitted to a variable-slope sigmoidal function using GraphPad Prism 6.0f to
59
generate IC50 values. All data were visually assessed for parasite growth above background
60
and integrity of curve fits.
61 62
Parasite susceptibility to DHA was also assessed using the new ex vivo ring-stage
63
survival assay (3). 20 μl erythrocyte pellets were added to 1.0 ml RPMI 1640 medium
64
supplemented with 25 mM HEPES, 0.2% NaHCO3, 0.1 mM hypoxanthine, 100 μg/ml
65
gentamicin, and 0.5% Albumax I (Invitrogen) containing 700 nM DHA or, for controls, 0.1%
66
DMSO at a hematocrit of 2%. Cultures were incubated at 37°C under 3% O2, 5% CO2, and
67
92% N2, and after 6 hours washed three times with 10 ml RPMI pre-warmed to 37°C, and
68
then placed in drug-free culture media. 72 hours after assay initiation, Giemsa-stained thin
69
smears were prepared. Cultures were considered viable and appropriate for assessment if
70
control parasitemia increased since culture initiation. Parasitemias in control cultures were
71
determined by counting parasites per 1000-2000 erythrocytes. In DHA-pulsed cultures,
72
10,000-20,000 cells were scanned for parasites. RSA survival rates were expressed as the
73
proportion of parasites in the DHA-treated cultures relative to controls, at the end of the 72
74
hour assay. For some samples, cultures were maintained for up to 4 weeks after initiation
75
of the experiment, with smears assessed on alternating days. For positive cultures, blood
76
spots were collected on filter paper.
4
77 78
DNA was extracted from filter paper blood spots using Chelex-100, and gene
79
fragments spanning loci of interest were amplified by nested PCR (10, 18). K13 propeller-
80
encoding domains (codons 440-726) were dideoxy sequenced as previously described (4).
81
To detect polymorphisms in pfcrt and pfmdr1, multiplex ligase detection reaction–
82
fluorescent microsphere assays were performed as previously described (19, 20).
83
Nucleotide sequence data are available in the GenBank database under accession numbers
84
KR055739-KR055804.
85 86
We collected 53 isolates from patients with malaria in May-July 2014. Giemsa-
87
stained thin smears from all blood samples demonstrated only P. falciparum at
88
parasitemias of 0.1-24.3%. We determined ex vivo IC50 data for 15 isolates. Values ranged
89
from 0.4-2.7 nM (geometric mean = 1.6 nM) for DHA and 9.8-1060 nM for chloroquine
90
(Table 1).
91 92
As standard drug sensitivity assays have not identified artemisinin resistance in
93
Asian isolates, we also assessed DHA susceptibility using the ex vivo ring-stage survival
94
assay (3). 10 isolates were lost to contamination or failed to grow. For the remaining 43
95
isolates, assessed 72 hours after culture initiation, most DHA-pulsed cultures contained
96
pyknotic forms, and only 3 of 43 demonstrated healthy-appearing parasites, all with
97
parasitemias ≤0.025%. Of the 3 positive cultures, a single parasite was detected per
98
~15,000 erythrocytes in 2 cultures, and 3 parasites were detected in the third (RSA
99
survival rates of 0.7 - 1.9%). All 43 untreated control cultures demonstrated increased
5
100
parasitemia and healthy-appearing parasites over the course of the assay. Because of the
101
challenges of quantifying extremely low parasitemias, we assessed the viability of 14 DHA-
102
pulsed cultures that did not demonstrate parasites after 72 hours. These cultures were
103
maintained for up to 30 days after initiation of the experiment. 12 of these cultures
104
demonstrated parasites, beginning 15-29 days after culture initiation; two cultures
105
remained negative at 30 days. Thus, we did not observe enhanced growth of DHA-pulsed
106
parasites after 72 hours in culture, arguing against the presence of the artemisinin-
107
resistant phenotype, although most cultures survived the DHA pulse.
108 109
Sequencing the K13-propeller domains from the 53 isolates revealed two
110
nonsynonymous mutations, S552C (mixed with the wild type allele) and A578S, in
111
propeller domains 2 and 4, respectively (Figure 1). Ten isolates that grew in culture 15-29
112
days after the DHA pulse all had wild type K13 sequences. Two of these samples were from
113
isolates that were positive by microscopy 72 hours after the DHA pulse. Considering the
114
putative drug transporters encoded by pfcrt and pfmdr1, polymorphism prevalences were
115
similar to those described recently from other regions of Uganda, with increased
116
prevalence of wild type sequences at pfcrt K76, pfmdr1 N86, and pfmdr1 D1246 compared
117
to older studies (Figure 1) (20, 21). Sensitivity to chloroquine correlated well with the pfcrt
118
K76T sequence, with IC50 values >120 nM for all mutant parasites,
