Donna Huber, Author at Center for Tropical and Emerging Global Diseases

Diagnostics to support elimination of lymphatic filariasis-Development of two target product profiles

Donna Huber on November 15, 2021

As lymphatic filariasis (LF) programs move closer to established targets for validation elimination of LF as a public health problem, diagnostic tools capable of supporting the needs of the programs are critical for success. Known limitations of existing diagnostic tools make it challenging to have confidence that program endpoints have been achieved. In 2019, the World Health Organization (WHO) established a Diagnostic Technical Advisory Group (DTAG) for Neglected Tropical Diseases tasked with prioritizing diagnostic needs including defining use-cases and target product profiles (TPPs) for needed tools. Subsequently, disease-specific DTAG subgroups, including one focused on LF, were established to develop TPPs and use-case analyses to be used by product developers. Here, we describe the development of two priority TPPs for LF diagnostics needed for making decisions for stopping mass drug administration (MDA) of a triple drug regimen and surveillance. Utilizing the WHO core TPP development process as the framework, the LF subgroup convened to discuss and determine attributes required for each use case. TPPs considered the following parameters: product use, design, performance, product configuration and cost, and access and equity. Version 1.0 TPPs for two use cases were published by WHO on 12 March 2021 within the WHO Global Observatory on Health Research and Development. A common TPP characteristic that emerged in both use cases was the need to identify new biomarkers that would allow for greater precision in program delivery. As LF diagnostic tests are rarely used for individual clinical diagnosis, it became apparent that reliance on population-based surveys for decision making requires consideration of test performance in the context of such surveys. In low prevalence settings, the number of false positive test results may lead to unnecessary continuation or resumption of MDA, thus wasting valuable resources and time. Therefore, highly specific diagnostic tools are paramount when used to measure low thresholds. The TPP process brought to the forefront the importance of linking use case, program platform and diagnostic performance characteristics when defining required criteria for diagnostic tools.

Kimberly Y Won, Katherine Gass, Marco Biamonte, Daniel Argaw Dagne, Camilla Ducker, Christopher Hanna, Achim Hoerauf, Patrick J Lammie, Sammy M Njenga, Rahmah Noordin, Kapa D Ramaiah, Reda Ramzy, Ronaldo G Carvalho Scholte, Anthony W Solomon, Ashley A Souza, Jordan Tappero, Emily Toubali, Gary J Weil, Steven A Williams, Jonathan D King. PLoS Negl Trop Dis 15(11): e0009968. https://doi.org/10.1371/journal.pntd.0009968

Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveals expanded transporter repertoire and duplication of entire chromosome ends including subtelomeric regions

Donna Huber on November 11, 2021

Cryptosporidiosis is a leading cause of waterborne diarrheal disease globally and an important contributor to mortality in infants and the immunosuppressed. Despite its importance, the Cryptosporidium community has only had access to a good, but incomplete, Cryptosporidium parvum IOWA reference genome sequence. Incomplete reference sequences hamper annotation, experimental design and interpretation. We have generated a new C. parvum IOWA genome assembly supported by PacBio and Oxford Nanopore long-read technologies and a new comparative and consistent genome annotation for three closely related species C. parvum, Cryptosporidium hominis and Cryptosporidium tyzzeri We made 1,926 C. parvum annotation updates based on experimental evidence. They include new transporters, ncRNAs, introns and altered gene structures. The new assembly and annotation revealed a complete Dnmt2 methylase ortholog. Comparative annotation between C. parvum, C. hominis and C. tyzzeri revealed that most “missing” orthologs are found suggesting that the biological differences between the species must result from gene copy number variation, differences in gene regulation and single nucleotide variants (SNVs). Using the new assembly and annotation as reference, 190 genes are identified as evolving under positive selection, including many not detected previously. The new C. parvum IOWA reference genome assembly is larger, gap free and lacks ambiguous bases. This chromosomal assembly recovers all 16 chromosome ends, 13 of which are contiguously assembled. The three remaining chromosome ends are provisionally placed. These ends represent duplication of entire chromosome ends including subtelomeric regions revealing a new level of genome plasticity that will both inform and impact future research.

Rodrigo P Baptista, Yiran Li, Adam Sateriale, Karen L Brooks, Alan Tracey, Mandy J Sanders, Brendan R E Ansell, Aaron R Jex, Garrett W Cooper, Ethan D Smith, Rui Xiao, Jennifer E Dumaine, Peter Georgeson, Bernard Pope, Matthew Berriman, Boris Striepen, James A Cotton, Jessica C Kissinger. Genome Res. 2021 Nov 11;gr.275325.121. doi: 10.1101/gr.275325.121.

Evolutionary genomics of APSE: a tailed phage that lysogenically converts the bacterium Hamiltonella defensa into a heritable protective symbiont of aphids

Donna Huber on November 10, 2021

Background: Most phages infect free-living bacteria but a few have been identified that infect heritable symbionts of insects or other eukaryotes. Heritable symbionts are usually specialized and isolated from other bacteria with little known about the origins of associated phages. Hamiltonella defensa is a heritable bacterial symbiont of aphids that is usually infected by a tailed, double-stranded DNA phage named APSE.

Methods: We conducted comparative genomic and phylogenetic studies to determine how APSE is related to other phages and prophages.

Results: Each APSE genome was organized into four modules and two predicted functional units. Gene content and order were near-fully conserved in modules 1 and 2, which encode predicted DNA metabolism genes, and module 4, which encodes predicted virion assembly genes. Gene content of module 3, which contains predicted toxin, holin and lysozyme genes differed among haplotypes. Comparisons to other sequenced phages suggested APSE genomes are mosaics with modules 1 and 2 sharing similarities with Bordetella-Bcep-Xylostella fastidiosa-like podoviruses, module 4 sharing similarities with P22-like podoviruses, and module 3 sharing no similarities with known phages. Comparisons to other sequenced bacterial genomes identified APSE-like elements in other heritable insect symbionts (Arsenophonus spp.) and enteric bacteria in the family Morganellaceae.

Conclusions: APSEs are most closely related to phage elements in the genus Arsenophonus and other bacteria in the Morganellaceae.

Bret M Boyd, Germain Chevignon, Vilas Patel, Kerry M Oliver, Michael R Strand. Virol J. 2021 Nov 10;18(1):219. doi: 10.1186/s12985-021-01685-y.

The transcriptome of Balamuthia mandrillaris trophozoites for structure-guided drug design

Donna Huber on November 4, 2021

Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a ‘brain-eating’ disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Current treatments of encephalitis due to B. mandrillaris lack efficacy, leading to case fatality rates above 90%. Using our recently published methodology to discover potential drugs against pathogenic amoebas, we screened a collection of 85 compounds with known antiparasitic activity and identified 59 compounds that impacted the growth of Balamuthia trophozoites at concentrations below 220 µM. Since there is no fully annotated genome or proteome of B. mandrillaris, we sequenced and assembled its transcriptome from a high-throughput RNA-sequencing (RNA-Seq) experiment and located the coding sequences of the genes potentially targeted by the growth inhibitors from our compound screens. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing. These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections.

Isabelle Q Phan, Christopher A Rice, Justin Craig, Rooksana E Noorai, Jacquelyn R McDonald, Sandhya Subramanian, Logan Tillery, Lynn K Barrett, Vijay Shankar, James C Morris, Wesley C Van Voorhis, Dennis E Kyle, Peter J Myler. Sci Rep. 2021 Nov 4;11(1):21664. doi: 10.1038/s41598-021-99903-8.

The Histidine Ammonia Lyase of Trypanosoma cruzi Is Involved in Acidocalcisome Alkalinization and Is Essential for Survival under Starvation Conditions

Donna Huber on November 2, 2021

Trypanosoma cruzi, the agent of Chagas disease, accumulates polyphosphate (polyP) and Ca²⁺ inside acidocalcisomes. The alkalinization of this organelle stimulates polyP hydrolysis and Ca²⁺ release. Here, we report that histidine ammonia lyase (HAL), an enzyme that catalyzes histidine deamination with production of ammonia (NH₃) and urocanate, is responsible for acidocalcisome alkalinization. Histidine addition to live parasites expressing HAL fused to the pH-sensitive emission biosensor green fluorescent protein (GFP) variant pHluorin induced alkalinization of acidocalcisomes. PolyP decreased HAL activity of epimastigote lysates or the recombinant protein but did not cause its polyphosphorylation, as determined by the lack of HAL electrophoretic shift on NuPAGE gels using both in vitro and in vivo conditions. We demonstrate that HAL binds strongly to polyP and localizes to the acidocalcisomes and cytosol of the parasite. Four lysine residues localized in the HAL C-terminal region are instrumental for its polyP binding, its inhibition by polyP, its function inside acidocalcisomes, and parasite survival under starvation conditions. Expression of HAL in yeast deficient in polyP degradation decreased cell fitness. This effect was enhanced by histidine and decreased when the lysine-rich C-terminal region was deleted. In conclusion, this study highlights a mechanism for stimulation of acidocalcisome alkalinization linked to amino acid metabolism.

IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and is characterized by the presence of acidocalcisomes, organelles rich in phosphate and calcium. Release of these molecules, which are necessary for growth and cell signaling, is induced by alkalinization, but a physiological mechanism for acidocalcisome alkalinization was unknown. In this work, we demonstrate that a histidine ammonia lyase localizes to acidocalcisomes and is responsible for their alkalinization.

Brian S Mantilla, Cristina Azevedo, Paul W Denny, Adolfo Saiardi, Roberto Docampo. mBio. 2021 Nov 2;e0198121. doi: 10.1128/mBio.01981-21.

Expeditious recruitment of circulating memory CD8 T cells to the liver facilitates control of malaria

Donna Huber on November 2, 2021

Circulating memory CD8 T cell trafficking and protective capacity during liver-stage malaria infection remains undefined. We find that effector memory CD8 T cells (Tem) infiltrate the liver within 6 hours after malarial or bacterial infections and mediate pathogen clearance. Tem recruitment coincides with rapid transcriptional upregulation of inflammatory genes in Plasmodium-infected livers. Recruitment requires CD8 T cell-intrinsic LFA-1 expression and the presence of liver phagocytes. Rapid Tem liver infiltration is distinct from recruitment to other non-lymphoid tissues in that it occurs both in the absence of liver tissue resident memory “sensing-and-alarm” function and ∼42 hours earlier than in lung infection by influenza virus. These data demonstrate relevance for Tem in protection against malaria and provide generalizable mechanistic insights germane to control of liver infections.

Mitchell N Lefebvre, Fionna A Surette, Scott M Anthony, Rahul Vijay, Isaac J Jensen, Lecia L Pewe, Lisa S Hancox, Natalija Van Braeckel-Budimir, Stephanie van de Wall, Stina L Urban, Madison R Mix, Samarcith P Kurup, Vladimir P Badovinac, Noah S Butler, John T Harty. Cell Rep. 2021 Nov 2;37(5):109956. doi: 10.1016/j.celrep.2021.109956.

VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center

Donna Huber on October 28, 2021

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) represents the 2019 merger of VectorBase with the EuPathDB projects. As a Bioinformatics Resource Center funded by the National Institutes of Health, with additional support from the Welllcome Trust, VEuPathDB supports >500 organisms comprising invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Designed to empower researchers with access to Omics data and bioinformatic analyses, VEuPathDB projects integrate >1700 pre-analysed datasets (and associated metadata) with advanced search capabilities, visualizations, and analysis tools in a graphic interface. Diverse data types are analysed with standardized workflows including an in-house OrthoMCL algorithm for predicting orthology. Comparisons are easily made across datasets, data types and organisms in this unique data mining platform. A new site-wide search facilitates access for both experienced and novice users. Upgraded infrastructure and workflows support numerous updates to the web interface, tools, searches and strategies, and Galaxy workspace where users can privately analyse their own data. Forthcoming upgrades include cloud-ready application architecture, expanded support for the Galaxy workspace, tools for interrogating host-pathogen interactions, and improved interactions with affiliated databases (ClinEpiDB, MicrobiomeDB) and other scientific resources, and increased interoperability with the Bacterial & Viral BRC.

Beatrice Amos, Cristina Aurrecoechea, Matthieu Barba, Ana Barreto, Evelina Y Basenko, Wojciech Bażant, Robert Belnap, Ann S Blevins, Ulrike Böhme, John Brestelli, Brian P Brunk, Mark Caddick, Danielle Callan, Lahcen Campbell, Mikkel B Christensen, George K Christophides, Kathryn Crouch, Kristina Davis, Jeremy DeBarry, Ryan Doherty, Yikun Duan, Michael Dunn, Dave Falke, Steve Fisher, Paul Flicek, Brett Fox, Bindu Gajria, Gloria I Giraldo-Calderón, Omar S Harb, Elizabeth Harper, Christiane Hertz-Fowler, Mark J Hickman, Connor Howington, Sufen Hu, Jay Humphrey, John Iodice, Andrew Jones, John Judkins, Sarah A Kelly, Jessica C Kissinger, Dae Kun Kwon, Kristopher Lamoureux, Daniel Lawson, Wei Li, Kallie Lies, Disha Lodha, Jamie Long, Robert M MacCallum, Gareth Maslen, Mary Ann McDowell, Jaroslaw Nabrzyski, David S Roos, Samuel S C Rund, Stephanie Wever Schulman, Achchuthan Shanmugasundram, Vasily Sitnik, Drew Spruill, David Starns, Christian J Stoeckert, Sheena Shah Tomko, Haiming Wang, Susanne Warrenfeltz, Robert Wieck, Paul A Wilkinson, Lin Xu, Jie Zheng. Nucleic Acids Res. 2021 Oct 28;gkab929. doi: 10.1093/nar/gkab929.

Control and elimination of lymphatic filariasis in Oceania: Prevalence, geographical distribution, mass drug administration, and surveillance in Samoa, 1998-2017

Donna Huber on October 27, 2021

Lymphatic filariasis (LF) is a major public health problem globally and in the Pacific Region. The Global Programme to Eliminate LF has made great progress but LF is persistent and resurgent in some Pacific countries and territories. Samoa remains endemic for LF despite elimination efforts through multiple two-drug mass drug administrations (MDA) since 1965, including renewed elimination efforts started in 1999 under the Pacific Programme for Elimination of LF (PacELF). Despite eight rounds of national and two rounds of subnational MDA under PacELF, Samoa failed transmission assessment surveys (TAS) in all three evaluation units in 2017. In 2018, Samoa was the first to distribute countrywide triple-drug MDA using ivermectin, diethylcarbamazine (DEC), and albendazole. This paper provides a review of MDAs and historical survey results from 1998 to 2017 in Samoa and highlights lessons learnt from LF elimination efforts, including challenges and potential ways to overcome them to successfully achieve elimination.

Patricia M Graves, Hayley Joseph, Shaun P Coutts, Helen J Mayfield, Fuatai Maiava, Tile Ann Ah Leong-Lui, Palanitina Tupuimatagi Toelupe, Vailolo Toeaso Iosia, Siatua Loau, Paulo Pemita, Take Naseri, Robert Thomsen, Alvaro Berg Soto, Thomas R Burkot, Peter Wood, Wayne Melrose, Padmasiri Aratchige, Corinne Capuano, Sung Hye Kim, Masayo Ozaki, Aya Yajima, Patrick J Lammie, Eric Ottesen, Lepaitai Hansell, Rasul Baghirov, Colleen L Lau, Kazuyo Ichimori. Adv Parasitol. 2021;114:27-73. doi: 10.1016/bs.apar.2021.03.002

Researchers implement new tool to screen drugs for treating malaria relapses

Donna Huber on October 21, 2021

Steve Maher, assistant research scientist in the Center for Tropical Emerging and Global Diseases, leads a team of researchers who have implemented a new screening tool to determine if a drug candidate kills hypnozoites, the cause of malaria relapses. (Photo by Donna Huber)

Globally, efforts to control malaria caused by Plasmodium vivax are lagging behind that of other species of Plasmodium due to its unique biology. A team of researchers at the University of Georgia, the Institute Pasteur of Cambodia, and Shoklo Malaria Research Unit in Thailand detail a new screening tool and report for the first time a method capable of discovering novel experimental drug compounds for use against vivax malaria. Their study was recently published in Scientific Reports.

The parasite species P. vivax is the most widespread cause of malaria. While not as deadly as malaria caused by P. falciparum, it can cause severe disease and has a significant impact on both national economies and personal finances, in part due to this species’ propensity to cause relapses.

Relapses are caused by hypnozoites, a form of the parasite residing in the liver, which can lie dormant for a period of time before causing another symptomatic blood infection. During this period of dormancy, hypnozoites are not susceptible to standard antimalarials, meaning a patient treated for a blood infection is not fully cured.

“With this assay, we can now tell earlier on in the drug discovery process if a compound is going to work against hypnozoites,” said Steven Maher, assistant research scientist at the Center for Tropical and Emerging Global Diseases and lead researcher on the study. “In this study, we were able to identify three new drugs that kill dormant hypnozoites.”

One of the drugs identified looks promising as a possible new treatment, though Maher said it will need more testing. The other two could be useful in studying hypnozoite biology and increase understanding of such things as the mechanisms of dormancy.

The team’s report also shows how two current antimalarial drugs, chloroquine and tafenoquine, synergistically work together to kill hypnozoites. However, these drugs cannot be administered to children and pregnant women (due to their known side effects), nor to people who lack the enzyme called G6PD. Up to 20% of the population in southeast Asia are G6PD deficient.

“The current drug therapies work well to treat the symptomatic blood stage of vivax malaria,” said Steven Maher. “However, in vivax malaria we need to eliminate hypnozoites to fully cure the patient, and for that we need new therapies.”

To compound the problem, typical mouse models used in malaria drug research can’t determine if the experimental compounds work against hypnozoites because the Plasmodium species that infects mice doesn’t produce them. Additionally, because the assays used as the first step in discovering potential new drug compounds focus on the blood stage of the parasite, researchers need a different kind of assay that will allow them to test these compounds on hypnozoites, which requires a stable culture of liver cells.

“It’s a challenge because you have to get samples from where the vivax malaria is endemic,” said Maher. “Liver cells don’t stay viable in culture for long, and these assays take eight days to show results. The assay itself is difficult to run, but we have a great team of researchers in Cambodia and Thailand that has really helped to make this possible.”

The team is continuing to build better tools to overcome the challenges drug discovery in P. vivax faces as they begin to test these drugs in animal models.

This story first appeared at UGA Research.

Application of SARS-CoV-2 serology to address public health priorities

Donna Huber on October 14, 2021

Background: Antibodies against SARS-CoV-2 can be detected by various testing platforms, but a detailed understanding of assay performance is critical.
Methods: We developed and validated a simple enzyme-linked immunosorbent assay (ELISA) to detect IgG binding to the receptor-binding domain (RBD) of SARS-CoV-2, which was then applied for surveillance. ELISA results were compared to a set of complimentary serologic assays using a large panel of clinical research samples.
Results: The RBD ELISA exhibited robust performance in ROC curve analysis (AUC> 0.99; Se=89%, Sp=99.3%). Antibodies were detected in 23/353 (6.5%) healthcare workers, 6/9 RT-PCR-confirmed mild COVID-19 cases, and 0/30 non-COVID-19 cases from an ambulatory site. RBD ELISA showed a positive correlation with neutralizing activity (p = <0.0001, R2 = 0.26).
Conclusions: We applied a validated SARS-CoV-2-specific IgG ELISA in multiple contexts and performed orthogonal testing on samples. This study demonstrates the utility of a simple serologic assay for detecting prior SARS-CoV-2 infection, particularly as a tool for efficiently testing large numbers of samples as in population surveillance. Our work also highlights that precise understanding of SARS-CoV-2 infection and immunity at the individual level, particularly with wide availability of vaccination, may be improved by orthogonal testing and/or more complex assays such as multiplex bead assays.

Amy C. Sherman, Teresa Smith, Yerun Zhu, Kaitlin Taibl, Jessica Howard-Anderson, Taylor Landay, Nora Pisanic, Jennifer Kleinhenz, Trevor W. Simon, Daniel Espinoza, Skyler Hammond, Nadine Rouphael, Huifeng Shen, Jessica K. Fairley, Jaime A. Cardona-Ospina, Alfonso J. Rodriguez-Morales, Lakshmanane Premkumar, Jens Wrammert, Rick Tarleton, Scott Fridkin, Christopher D. Heaney, Erin M. Scherer and Matthew H. Collins. Frontiers in Public Health, Oct. 2021, doi: 10.3389/fpubh.2021.744535

Author: Donna Huber