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.

Targeting the artemisinin resistant malaria by repositioning of the anti-Hepatitis C Virus drug Alisporivir

The rapid emergence of P. falciparum-resistant strains raises an urgent need to find new antimalarial drug candidates. This study reports the rational repositioning of the anti-Hepatitis C Virus drug, Alisporivir, a non-immunosuppressive analog of cyclosporin A (CsA) against multiple, drug-resistant strains of P. falciparum. Alisporivir being non-hemolytic has been proven to be a better drug than CsA. Indeed, our study also demonstrated the same. Alisporivir inhibited chloroquine-sensitive parasite growth with an IC50 of 196.6nM. Alisporivir also inhibited the growth of chloroquine-resistant parasites with an IC50 of 422.1nM. Alisporivir exhibited, anti-malarial activity in in vivo. Further, we exploited the Cyclophilins targeting potential of Alisporivir against artemisinin-resistant malaria parasite owing to the fact that PfCyP-19B is one of the genes that is overexpressed in artemisinin-resistant parasite revealed by a population transcriptomic study. Our semiquantitative real-time transcript and immunofluorescence analysis confirmed the overexpression of PfCyP-19B in Artemisinin-resistant P. falciparum (PfKelch13R539T). Artemisinin resistance is attributed to slow clearance of ring stage parasites. Ring survival assay (RSA) is designed to access the potency of compounds on these dormant slow clearing parasites leading to drug resistance. Thus, the potency of Alisporivir against PfKelch13R539T was evaluated by RSA. A 2.5-fold decrease in parasite survival was detected with Alisporivir. Further, combination of Alisporivir with DHA found to potentiate the efficacy of DHA by 4.55-fold. These results support the hypothesis that targeting of resistance mechanism is a potential approach to deal with resistant parasite. Overall, this study demonstrates the rational reposition of Alisporivir against resistant malaria resistance.

Status of anthelmintic resistance of gastrointestinal nematodes in organized sheep and goat farms

Anthelmintic resistance has become a global problem that threatens livestock production worldwide. The present study was investigated the status of anthelmintic resistance in gastrointestinal (GI) nematodes of small ruminants in two organized sheep and goat farms in two different areas of Bangladesh by fecal egg count reduction test (FECRT) for albendazole, levamisole and ivermectin. In each farms, naturally infected animals were divided into four groups of 10 animals. Fecal samples from each group were collected on day 0 and day 14 of post treatment to measure the eggs per gram of feces (EPG). The fecal samples of each group before and after treatment were also considered for culture to identify resistant parasite. In case of organized sheep farm, the result of FECRT of albendazole was 90.17, 95% confidence with upper and lower limit was 97.82 and 55.68, respectively. The result of FECRT of levamisole and ivermectin was 98.25 and 96.77, 95% confidence with upper and lower limit was 99.79, 85.12 and 99.11, 88.31, respectively. In case of organized goat farm, the result of FECRT of albendazole was 100, 95% confidence with upper and lower limit was 0 and 0, respectively. The result of FECRT of levamisole and ivermectin was 97.99 and 100, 95% confidence with upper and lower limit was 99.59, 90.28 and 0, 0 respectively. The results revealed that gastrointestinal nematodes were found to be resistant to albendazole in organized sheep farm and suspected to be resistant to levamisole and ivermectin. In organized goat farms, the GI nematodes found to be susceptible to all anthelmintics used for this study. Coproculture revealed that Haemonchus spp. were resistant parasite in sheep farm. This seems to be the first documentation of anthelmintic resistance against GI nematodes in organized sheep and goat farms in Bangladesh. Appropriate measures need to be taken to overcome the situation. Asian J. Med. Biol. Res. December 2018, 4(4): 378-382

Amodiaquine resistance in Plasmodium berghei is associated with PbCRT His95Pro mutation, loss of chloroquine, artemisinin and primaquine sensitivity, and high transcript levels of key transporters

Background: The human malaria parasite Plasmodium falciparum has evolved complex drug evasion mechanisms to all available antimalarials. To date, the combination of amodiaquine-artesunate is among the drug of choice for treatment of uncomplicated malaria. In this combination, a short acting, artesunate is partnered with long acting, amodiaquine for which resistance may emerge rapidly especially in high transmission settings. Here, we used a rodent malaria parasite Plasmodium berghei ANKA as a surrogate of P. falciparum to investigate the mechanisms of amodiaquine resistance. Methods: We used serial technique to select amodiaquine resistance by submitting the parasites to continuous amodiaquine pressure. We then employed the 4-Day Suppressive Test to monitor emergence of resistance and determine the cross-resistance profiles. Finally, we genotyped the resistant parasite by PCR amplification, sequencing and relative quantitation of mRNA transcript of targeted genes. Results: Submission of P. berghei ANKA to amodiaquine pressure yielded resistant parasite within thirty-six passages. The effective dosage that reduced 90% of parasitaemia (ED90) of sensitive line and resistant line were 4.29mg/kg and 19.13mg/kg, respectively. After freezing at -80ºC for one month, the resistant parasite remained stable with an ED90 of 18.22mg/kg. Amodiaquine resistant parasites are also resistant to chloroquine (6fold), artemether (10fold), primaquine (5fold), piperaquine (2fold) and lumefantrine (3fold). Sequence analysis of Plasmodium berghei chloroquine resistant transporter revealed His95Pro mutation. No variation was identified in Plasmodium berghei multidrug resistance gene-1 (Pbmdr1), Plasmodium berghei deubiquitinating enzyme-1 or Plasmodium berghei Kelch13 domain nucleotide sequences. Amodiaquine resistance is also accompanied by high mRNA transcripts of key transporters; Pbmdr1, V-type/H+ pumping pyrophosphatase-2 and sodium hydrogen ion exchanger-1 and Ca2+/H+ antiporter. Conclusions: Selection of amodiaquine resistance yielded stable “multidrug-resistant’’ parasites and thus may be used to study common resistance mechanisms associated with other antimalarial drugs. Genome wide studies may elucidate other functionally important genes controlling AQ resistance in P. berghei.