Artemisinin combination therapy fails even in the absence of Plasmodium falciparum kelch13 gene polymorphism in Central India

Artemisinin is the frontline fast-acting anti-malarial against P. falciparum. Emergence and spread of resistant parasite in eastern-India poses a threat to national malaria control programs. Therefore, the objective of our study is to evaluate the artesunate-sulfadoxine-pyrimethamine efficacy in Central India. 180 monoclonal P. falciparum-infected patients received standard ASSP therapy during August 2015–January 2017, soon after diagnosis and monitored over next 42-days. Artemisinin-resistance was assessed through in-vivo parasite clearance half-life (PC1/2), ex-vivo ring-stage survivability (RSA), and genome analysis of kelch13 and other candidate gene (pfcrt, pfmdr1, pfatpase 6, pfdhfr and pfdhps). Of 180 P. falciparum positive patients, 9.5% showed increased PC1/2 (> 5.5 h), among them eleven isolates (6.1%) showed reduced sensitivity to RSA. In 4.4% of cases, parasites were not cleared by 72 h and showed prolonged PC1/2(5.6 h) (P < 0.005) along with significantly higher RSA (2.2%) than cured patients (0.4%). None of day-3 positive isolates contained the pfkelch13 mutation implicated in artemisinin resistance. Parasite recrudescence was observed in 5.6% patients, which was associated with triple dhfr–dhps (A16I51R59N108I164–S436G437K540G581T613) combination mutation. Emergence of reduced sensitivity to artesunate-sulfadoxine-pyrimethamine, in central India highlighted the risk toward spread of resistant parasite across different parts of India. Day-3 positive parasite, featuring the phenotype of artemisinin-resistance without pfkelch13 mutation, suggested kelch13-independent artemisinin-resistance.

Plasma availability of dihydroartemisinin. We measured artemisinin exposure in patients through detection of plasma DHA. Mean plasma DHA was recorded as 4052 nM, (95% CI 3925. .1) at 1.5 h and 2137. 5  ACT efficacy. We observed persistence of parasite after 72 h of ASSP exposure in 9 patients (5%), with high median axillary temperature of 38 °C (95% CI 37.8-38.2 °C), corresponding patients also showed prolonged median PC 1/2 of 5.6 h (95% Cl 5.5-5.7 h). Among them, 8 patients represented adequate plasma DHA level. Of them, 7 patients showed reduced sensitivity to DHA (in vitro) and designated as early ACT failure cases.

Molecular characterization of parasite recrudescence.
After PCR corrections, we identified 10 cases of recrudescence. The mean parasite load on the day of recrudescence was found 8285/µl (95% CI 2726-13,843). The mean body temperature on the day of recrudescence was very high 38.6 °C (95% CI 38.2-39.0 °C). We observed 3 cases of parasite recrudescence within day-7 and another 7 cases between day-8 to day 42, corresponding isolates representing significantly higher IC 50 s for sulfadoxine and pyrimethamine. Parasite recrudescence was the true cause of late ASSP failure. We noticed combination of triple dhfr and dhps mutation (A 16 I 51 R 59 N 108 I 164 -S 436 G 437 K 540 G 581 T 613 ) was highly correlated with parasite recrudescence (P < 0.01), while A 16 N 51 R 59 N 108 I 164 -S 436 G 437 K 540 G 581 T 613 and A 16 I 51 C 59 N 108 I 164 -A 436 G 437 K 540 A 581 A 613 mutations also contributed crucial role in parasite recrudescence ( mutations exhibited moderate to high IC 50 for pyrimethamine and sulfadoxine but never connected with recrudescence (P = 0.73) (Fig. 3). On the day of parasite recurrence, all the LTFs received a standard dose of AMLF and all showed treatment success after 42 days follow-up (Table 6).

Discussion
Emergence and spreading of partial artemisinin resistant parasites in eastern India 27,28 along with late ACT failures in north-east India 34 called for a systematic screening of ASSP in central India, as the second highest number of malarial infections was reported from the state of Chhattisgarh. We found the day-3 positive parasite with prolonged parasite clearance half-life (> 5.5 h), along with recrudescence cases. 9.4% of patients showed Figure 2. (A) Proportion of Parasite clearance phenotypes: we had classified four different parasite clearance phenotypes depending on the parasite clearance time. Parasites, those who cleared within 36 h of drug administration were classified as Rapid clearing parasite (RCP) whereas parasites cleared by 48 h of drug exposure were designated as Parasite clearance normal (PCN). In patients, those whose parasites were cleared by > 48 h to ≥ 72, were designated as Delayed clearing parasite (DCP). Parasites were not cleared after 72 h of drug exposure were designated as Very slow clearing parasite (VSCP). (B) Frequency of different Parasite clearance phenotypes in relation to PC 1/2 : Parasite clearance normal (PCN) phenotype (40%), was most prevalent followed by delayed clearing parasite (DCP; 28.88%) and rapid clearing parasite (RCP; 26.11%). Interestingly, 5% of isolates represented VSCP phenotypes. We found low median PC 1/2 in RCP (2.6 h) and PCN phenotype (2.8 h). Higher PC 1/2 was observed in DCP phenotype (4.2 h) while very high median PC 1/2 was recorded in VSCP phenotype (5.6 h) which proved these phenotypes perhaps less sensitive to ASSP therapy.        Here PYR and SDX respectively stand for "pyrimethamine, and sulfadoxine. " The blue line (corresponding to 2000 nM of PYR) represented the in vitro PYR resistance, while the red line (corresponding to 3000 nM of SDX) represented the in vitro SDX resistance. We observed prevalence of SP resistant parasites. The isolates presenting triple dhfr and dhps mutation (AIRNI-SGKGT; ANRNI-SGKGT) and double dhfr and dhps combination mutation (AICNI-AGKAA) represented very high IC 50 for pyrimethamine and sulfadoxine and proved to be highly resistant to PYR and SDX (P < 0.01). Isolates contained ANRNI-AGKAA, AICNI-SGKAA, and ANRNI-AAKAA mutations exhibited moderate to high IC 50 for pyrimethamine and sulfadoxine but never connected with recrudescence (P = 0.73). Pyrimethamine sensitive and sulfadoxine resistant 3D7 strain was used as a control strain. www.nature.com/scientificreports/ prolonged PC 1/2 (> 5.5 h) which was an alarming sign. Prolonged parasite clearance was an indicator of decreased efficacy of fast acting artemisinin within ACT which perhaps led to ACT failure 12,16,19 . We observed the median PC 1/2 of very slow-clearing parasite (VSCP) (5.6 h) and delayed-clearing parasite (4.2 h) phenotype were significantly higher from the PC 1/2 of Thailand-Myanmar border (3.7 h) and lower from Western Cambodia (5.9 h) 16 . Identification of these VSCP parasite phenotypes, confirmed the emergence of parasites that became less sensitive to artemisinin in vivo 12 . In spite of this, we also observed isolates with reduced sensitivity to DHA in vitro. The corresponding isolates of VSCP phenotype (5%) represented higher RSA (0-3 h) (2.2%) than the cured patients (0.46%), which proved those VSCP phenotypes were less sensitive to artemisinin in vitro. Previous reports from Thailand suggested that increased viability of ring-stage parasites (RSA (0-3 h) > 1%) was strongly associated with elevated PC 1/2 16 . Recent reports suggested that apart from reduced efficacy of fast acting artemisinin drugs, failure in late-acting combinations also contributes towards the increment of PC 1/2 14 . Thus, the reduced artemisinin sensitivity along with elevated PC 1/2 of the ring stage parasite resulted in reduced sensitivity to ASSP combination therapy. However, most studies in India reported no evidence of less susceptibility to artemisinin in vivo or in vitro [34][35][36] . Some recent studies reported the spreading of parasites with reduced sensitivity to artemisinin in vivo in eastern India 27,28 . We have identified 9, day-3 positive cases; among them, 7 (3.9%) patients were confirmed as ETF whereas 10 (5.6%) patients were identified as true recrudescence (LTF) cases (Table 2). Given the results of our study, although the numbers of treatment failure cases were not very high, we confirmed the emergence of reduced susceptibility to ASSP combination in this part of India. Nevertheless, findings of reduced efficacy of ASSP in Central India suggested the possibility of emergence of resistant parasites in the near future.

dhfr-dhps combination haplotype
ETF generally occurs due to reduced efficacy of fast-acting artemisinin in combination therapy 18 . The mechanism behind the reduced susceptibility to artemisinin is not fully clear, but genome wide analyses, ex-vivo RSA, and transfection studies suggested that artemisinin resistance is predominantly related to pfkelch13 gene polymorphism 14,[18][19][20][21][22] . Interestingly, predominance of wild pfkelch13 allele was observed in this parasite population of Central India. Despite the direct correlation with pfkelch13 polymorphism, we had identified 7 (3.9%) ETF cases with prolonged PC 1/2 as well as increased ex-vivo RSA (0-3 h) survivability, without pfkelch13 polymorphism. However, the findings of our study were quite uncommon. Our observations from this study suggest several novel aspects of artemisinin-resistance. Firstly, the kelch13-indpendent artemisinin-resistance which was previous reported only in Thailand 24,37,38 . Although, we identified 3 isolates with pfkelch13-A675V polymorphism which showed positive association with increased ex-vivo RSA (0-3 h) , but not with treatment outcome like previous reports from in north-east India and southern Rwanda 39,40 . The results do not represent outliers, as we studied a statistically valid number of patients. Secondly, the absence of pfkelch13 polymorphism suggests definitive roles for other genetic factors in reduced artemisinin-sensitivity and emergence of artemisinin-resistance. Of note, polymorphism in Ca 2+ ATPase6 gene (pfatpase6) had some role in reducing susceptibility to artemisinin 22 . However, the likelihood of this mechanism has been debated 41 . Likening the observations from Thai-Cambodian border 12 , we have not so far found any definitive correlation between pfatpase6 mutation and ASSP efficacy In India. Thirdly, pfcrt, pfmdr, and pffd polymorphisms plausibly represent the genetic background required for the onset of pfkelch13 polymorphism, as mutations in those genes showed strong association with the beginning of pfkelch13 mutation during selection of artemisinin-resistance 18,23,27,28 . Indeed, in our study, day-3 positive cases demonstrated acquired mutations in pfcrt (K76T, I356T) and pfmdr1 (N86Y, Y184F) genes similar to the findings reported in eastern and north-India 27,37 . A recent report showed P. falciparum strains in South-East Asia having some genetic attenuation to develop novel mutations that caused artemisinin-resistance 42 . Fourthly, the precise genetic architectures in relation to reduced artemisinin-sensitivity in the East, North-East, South-West, and Central India are plausibly different owing to extensive variations in socio-demographic, environmental, seasonal and parasite-vector relationships 26,27,39 .
We detected 10 recrudescence cases in our study. The clinical manifestations of recrudescence were related with less susceptibility of longer acting partner drug (SP), as evidence, parasite isolates showed acquired combination mutation in pfdhfr-pfdhps gene 8,27,30 . Isolates representing sextuple or quintuple dhfr-dhps combination mutations (A 16 I 51 R 59 N 108 I 164 -S 436 G 437 K 540 G 581 T 613 , A 16 N 51 R 59 N 108 I 164 -S 436 G 437 K 540 G 581 T 613 and A 16 I 51 C 59 N 108 I 164 -A 436 G 437 K 540 A 581 A 613 ), exhibited very high IC 50 s for pyrimethamine and sulfadoxine, proving true resistance towards SP. Quadruple dhfr-dhps combination mutations with reduced SP sensitivity was previously reported from Chhattisgarh 29 . Despite the high prevalence of molecular markers associated with SP resistance, treatment failure rate especially LTFs were much less in number. The sensitivity of artesunate over this parasite population was still very high. Artemisinin derivatives within the ASSP were therefore able to kill most of the parasite and reduce the burden of partner drugs. That is how all treatment failure cases recovered after AMLF therapy suggesting that a standard six-dose-AMLF could be a potential second-line treatment against three-dose ASSP failure.
In conclusion, emergence of reduced sensitivity to artemisinin and predominance of SP resistant parasites suggested us for the evaluation of national drug policy as artemether-lumefantrine could be successor of ASSP. Finally, a National Malaria Eradication Program requires urgently for centralized and synchronized implementation of new drug-combinations and tracking of genetic mutations that might lead to higher level of resistance to artemisinin and its partner drug.

Methods
Study population. We conducted the study at Bhilai (21.21° N, 81.38° E) and Durg (21.19° N, 81.28° E), districts of Chhattisgarh, India during August 2015-January 2017. Chhattisgarh had contributed the second highest malaria incidence in 2014 9 . The inclusion criteria were age ≥ 3 years, axillary temperature ≥ 37.5 °C, shivering, headache during the past two 2 days or more, and no recent history of anti-malarial medication. All participants were screened for P. falciparum infection by microscopic examination of Giemsa-stained thick and www.nature.com/scientificreports/ thin blood smears. Quantification of parasitaemia was performed by counting the number of parasites per 8000 WBC. We usually count at least 200 fields using 100× oil immersion objectives. Every field generally comprises 4-5 WBCs. Parasites/µl blood was determined as (parasites/WBCs) × 8000 (WBC count/µl of blood). The minimum detectable parasitemia was 40 parasites/µl of blood. pfmspI (MAD20 and K1) and pfmspII (3D7 and FC27) alleles were screened to detect the clonality of infection. Patients having signs and symptoms of severe malaria, pregnant women, infants and poly-clonal P. falciparum infections were excluded 43 . The experimental design and protocols were duly approved by Vidyasagar University, Human ethical committee (VU/HEC 15-0051). We strictly followed the WHO and WWARN (WorldWide Anti-malarial Resistance Network) guideline along with the Helsinki protocol. We obtained duly signed informed consents from each patient and patient's guardian for minor (child patient) ( Fig. 1 for patient selection details).
ASSP efficacy. Those patients who fulfilled the inclusion criteria received the quality assured standard ASSP dose (supplied by Ministry of Health and Family-Welfare) of 4 mg/kg body weight artesunate once daily for 3 days and a single dose of 25 mg/kg body weight sulfadoxine along with 1.25 mg/kg bodyweight pyrimethamine on first day, under the supervision of medical officer 9 . Trained microscopists checked the thin blood smears at an interval of 6 h, until the patients became parasite free. We estimated the PC 1/2 by parasite clearance estimator 15 .
Patients who developed renal failure and those who vomited the drug were withdrawn from the study and sent for further care. We performed the follow-up evaluations on day 7, 14, 21, 28, 35, and 42 days after initial therapy, and responses were classified accordingly 43 . We performed unscheduled follow-up when symptoms of malaria reappear. Patients, not responding to ASSP, received artemether-lumefantrine rescue therapy (6 tablets, each containing 40 mg AM and 240 mg LF) and were further followed-up for next 42 days.
Plasma artemisinin testing. We collected 500 µl of intra-venous blood just before, at 1.5 h and 3 h (± 30 min) after initiation of ASSP therapy. We obtained plasma samples immediately and stored at -20 °C. We evaluated the plasma dihydro-artemisinin (DHA) to validate the sporadic drug exposure by liquid chromatography as stated previously 44 .
In vitro drug sensitivity testing. We adapted P. falciparum clinical isolates in vitro as described previously 28,45 . After culture adaptation parasites were allowed to proliferate for 72 h before doing the antimalarial drug (sulfadoxine, pyrimethamine, and chloroquine) exposure. We performed sensitivity testing of anti-malarial by hypoxanthine incorporation assay in triplicate, according to our standard laboratory protocol 46,47  Ring-stage survival testing. We performed ring-stage survival (RSA) assay in triplicate after culture adaption of clinical isolates as described earlier 17 . We treated 0-3 h post-invasive, highly synchronized early ring-stage parasites with 700nMoles of dihydro-artemisinin for 6 h, followed by washing with RPMI-1640 for three times and further cultivated for another 66 h. We measured parasite survival rates by microscopic examination of 10,000 RBCs per treatment replicate in Giemsa-stained thin blood smears.
Statistical analysis. We expressed our data as a univariate median; mean ± SEM. Fisher's exact test along with regression analyses were performed to correlate the treatment efficacies with molecular genotyping. We used the Clopper-Pearson method to calculate the 95% confidence intervals. Data were compared between two groups by Mann-Whitney U-test while Kruskal-Wallis-test was used to compare among more than two groups. We considered p < 0.05 statistically significant. All statistical analyses were performed through Graph Pad in-Stat 3.0 and Origin 6.1.