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In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites.

ABSTRACT: The unique relapsing nature of Plasmodium vivax infection is a major barrier to malaria eradication. Upon infection, dormant liver-stage forms, hypnozoites, linger for weeks to months and then relapse to cause recurrent blood-stage infection. Very little is known about hypnozoite biology; definitive biomarkers are lacking and in vitro platforms that support phenotypic studies are needed. Here, we recapitulate the entire liver stage of P. vivax in vitro, using a multiwell format that incorporates micropatterned primary human hepatocyte co-cultures (MPCCs). MPCCs feature key aspects of P. vivax biology, including establishment of persistent small forms and growing schizonts, merosome release, and subsequent infection of reticulocytes. We find that the small forms exhibit previously described hallmarks of hypnozoites, and we pilot MPCCs as a tool for testing candidate anti-hypnozoite drugs. Finally, we employ a hybrid capture strategy and RNA sequencing to describe the hypnozoite transcriptome and gain insight into its biology.

SUBMITTER: Gural N

PROVIDER: S-EPMC8048090 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites.

Gural Nil N Mancio-Silva Liliana L Miller Alex B AB Galstian Ani A Butty Vincent L VL Levine Stuart S SS Patrapuvich Rapatbhorn R Desai Salil P SP Mikolajczak Sebastian A SA Kappe Stefan H I SHI Fleming Heather E HE March Sandra S Sattabongkot Jetsumon J Bhatia Sangeeta N SN

Cell host & microbe 20180222 3

The unique relapsing nature of Plasmodium vivax infection is a major barrier to malaria eradication. Upon infection, dormant liver-stage forms, hypnozoites, linger for weeks to months and then relapse to cause recurrent blood-stage infection. Very little is known about hypnozoite biology; definitive biomarkers are lacking and in vitro platforms that support phenotypic studies are needed. Here, we recapitulate the entire liver stage of P. vivax in vitro, using a multiwell format that incorporates ...[more]

PMID: 29478773

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Project description:BackgroundThe screening of lead compounds against in vitro parasite cultures is an essential step in the development of novel anti-malarial drugs, but currently relies on laboratory parasite lines established in vitro during the last century. This study sought to establish in continuous culture a series of recent Plasmodium falciparum isolates to represent the current parasite populations in Africa, all of which are now exposed to artemisinin combination therapy.MethodsPre-treatment P. falciparum isolates were obtained in EDTA, and placed into continuous culture after sampling of DNA. One post-treatment blood sample was also collected for each donor to monitor parasite clonality during clearance in vivo. IC?? estimates were obtained for 11 anti-malarial compounds for each established parasite line, clonal multiplicity measured in vivo and in vitro, and polymorphic sites implicated in parasite sensitivity to drugs were investigated at the pfmdr1, pfcrt, pfdhfr, pfdhps and pfap2mu loci before and after treatment, and in the cultured lines.ResultsPlasmodium falciparum isolates from seven malaria patients with recent travel to three West African and two East African countries were successfully established in long-term culture. One of these, HL1211, was from a patient with recrudescent parasitaemia 14 days after a full course of artemether-lumefantrine. All established culture lines were shown to be polyclonal, reflecting the in vivo isolates from which they were derived, and at least two lines reliably produce gametocytes in vitro. Two lines displayed high chloroquine IC?? estimates, and carried the CVIET haplotype at codons 72-76, whereas the remaining five lines carried the CVMNK haplotype and were sensitive in vitro. All were sensitive to the endoperoxides dihydroartemisinin and OZ277, but IC?? estimates for lumefantrine varied, with the least sensitive parasites carrying pfmdr1 alleles encoding Asn at codon 86.ConclusionsThis study describes the establishment in continuous culture, in vitro drug sensitivity testing and molecular characterization of a series of multiclonal P. falciparum isolates taken directly from UK malaria patients following recent travel to various malaria-endemic countries in Africa. These "HL" isolates are available as an open resource for studies of drug response, antigenic diversity and other aspects of parasite biology.

Project description:Malaria is one of the most significant tropical diseases, and of the Plasmodium species that cause human malaria, P. vivax is the most geographically widespread. However, P. vivax remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous P. vivax in vitro culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term P. vivax culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for in vitro propagation. Reliance on very heterogeneous P. vivax field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined Aotus-adapted P. vivax Sal-1 strain to empirically evaluate different short-term in vitro culture conditions and compare them with previous reported attempts at P. vivax in vitro culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that P. vivax may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of Aotus-adapted Sal-1 for development of P. vivax laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development.

Project description:Plasmodium vivax is the most prevalent human malaria parasite and is likely to increase proportionally as malaria control efforts more rapidly impact the prevalence of Plasmodium falciparum. Despite the prominence of P. vivax as a major human pathogen, vivax malaria qualifies as a neglected and under-studied tropical disease. Significant challenges bringing P. vivax into the laboratory, particularly the capacity for long-term propagation of well-characterized strains, have limited the study of this parasite's red blood cell (RBC) invasion mechanism, blood-stage development, gene expression, and genetic manipulation.Patient isolates of P. vivax have been collected and cryopreserved in the rural community of Ampasimpotsy, located in the Tsiroanomandidy Health District of Madagascar. Periodic, monthly overland transport of these cryopreserved isolates to the country's National Malaria Control Programme laboratory in Antananarivo preceded onward sample transfer to laboratories at Case Western Reserve University, USA. There, the P. vivax isolates have been cultured through propagation in the RBCs of Saimiri boliviensis. For the four patient isolates studied to-date, the median time interval between sample collection and in vitro culture has been 454 days (range 166-961 days). The median time in culture, continually documented by light microscopy, has been 159 days; isolate AMP2014.01 was continuously propagated for 233 days. Further studies show that the P. vivax parasites propagated in Saimiri RBCs retain their ability to invade human RBCs, and can be cryopreserved, thawed and successfully returned to productive in vitro culture.Long-term culture of P. vivax is possible in the RBCs of Saimiri boliviensis. These studies provide an alternative to propagation of P. vivax in live animals that are becoming more restricted. In vitro culture of P. vivax in Saimiri RBCs provides an opening to stabilize patient isolates, which would serve as precious resources to apply new strategies for investigating the molecular and cellular biology of this important malaria parasite.

Project description:This review article aims to investigate the genotypic profiles of Plasmodium falciparum and Plasmodium vivax isolates collected across a wide geographic region and their association with resistance to anti-malarial drugs used in Indonesia. A systematic review was conducted between 1991 and date. Search engines, such as PubMed, Science Direct, and Google Scholar, were used for articles published in English and Indonesian to search the literature. Of the 471 initially identified studies, 61 were selected for 4316 P. falciparum and 1950 P. vivax individual infections. The studies included 23 molecular studies and 38 therapeutic efficacy studies. K76T was the most common pfcrt mutation. K76N (2.1%) was associated with the haplotype CVMNN. By following dihydroartemisinin-piperaquine (DHA-PPQ) therapy, the mutant pfmdr1 alleles 86Y and 1034C were selected. Low prevalence of haplotype N86Y/Y184/D1246Y pfmdr1 reduces susceptibility to AS-AQ. SNP mutation pvmdr1 Y976F reached 96.1% in Papua and East Nusa Tenggara. Polymorphism analysis in the pfdhfr gene revealed 94/111 (84.7%) double mutants S108N/C59R or S108T/A16V in Central Java. The predominant pfdhfr haplotypes (based on alleles 16, 51, 59,108, 164) found in Indonesia were ANCNI, ANCSI, ANRNI, and ANRNL. Some isolates carried A437G (35.3%) or A437G/K540E SNPs (26.5%) in pfdhps. Two novel pfdhps mutant alleles, I588F/G and K540T, were associated with six pfdhps haplotypes. The highest prevalence of pvdhfr quadruple mutation (F57L/S58R/T61M/S117T) (61.8%) was detected in Papua. In pvdhps, the only polymorphism before and after 2008 was 383G mutation with 19% prevalence. There were no mutations in the pfk13 gene reported with validated and candidate or associated k13 mutation. An increased copy number of pfpm2, associated with piperaquine resistance, was found only in cases of reinfection. Meanwhile, mutation of pvk12 and pvpm4 I165V is unlikely associated with ART and PPQ drug resistance. DHA-PPQ is still effective in treating uncomplicated falciparum and vivax malaria. Serious consideration should be given to interrupt local malaria transmission and dynamic patterns of resistance to anti-malarial drugs to modify chemotherapeutic policy treatment strategies. The presence of several changes in pfk13 in the parasite population is of concern and highlights the importance of further evaluation of parasitic ART susceptibility in Indonesia.

Dataset Information

In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites.

Publications

In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites.

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