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
Schistosomiasis, caused by several species of the parasitic worm Schistosoma, is a disease of poverty. The debilitating illness keeps people in a cycle of poverty due to missed educational and employment opportunities. In children, repeated infections often lead to anemia, malnutrition and learning disabilities. While there are no current preventive drug therapies or vaccines, annual mass drug administration (MDA) is often used to treat all elementary school children in countries where Schistosoma species are endemic.
“Treating with praziquantel through MDA programs is really successful in reducing schistosomiasis,” said Dan Colley, professor emeritus in the Franklin College of Arts and Sciences’ Department of Microbiology and former director of the Center for Tropical and Emerging Global Diseases. “However, in our studies we were seeing a few villages that didn’t really respond to annual MDA with lower numbers of infections. And it wasn’t just a few villages—in any given group of 25 study villages, we saw what we’re calling ‘persistent hotspots.’”
Schistosoma parasites develop in certain species of freshwater snails and are shed into the water where the form of the parasite that infects people can survive up to 24 hours. When an individual comes to the water to bathe, wash clothes, work or play, the parasites invade through the skin.
Several weeks after infection, the worms mature and begin to produce eggs that travel to the person’s bladder or intestines, where they are expelled through urine or stool. When freshwater sources are contaminated with human urine or fecal matter that contain these eggs, the life cycle begins again when the parasite eggs hatch and infect appropriate freshwater snails.
In a recent study published in PLOS ONE, Colley and his colleagues at the University of Georgia, Kenya Medical Research Institute (KEMRI), and Kenya’s Ministry of Health found high use of surface water and low use of latrines were factors associated with being a PHS and likely contributed to persistent levels of Schistosoma infection in these PHS villages—even in the face of annual MDA.
The study in western Kenya included both villages responding well to MDA (with a declining level of schistosomiasis) and those that are considered persistent hotspots. All the villages have fewer than 2,000 people, and most range between 500 and 1,500 residents. They often contain a crossroads that serves as the town center with a few shops. While the town center and the nearby houses have electricity, homes farther out might not. Villages considered PHS were located closer to open water sources, and more residents were employed in water-based jobs such as car washing and sand harvesting.
It was also found that these PHS villages had fewer latrines than villages that responded well to MDA, which likely led to more human fecal matter washing into the surface waters. The researchers believe that for these PHS villages, increasing latrine availability and use would reduce Schistosoma prevalence. However, it isn’t just a case of “if you build it, they will come.”
There are several barriers that would need to be overcome. Latrines, commonly called outhouses, are often smelly and attract flies and rats, which then attract snakes. In addition to issues of maintenance, there are social and educational components that need to be considered to change practices. But even when the people are willing, increased latrine use doesn’t always work.
Colley recounts the story of a group of car washers he and other colleagues at KEMRI and the Centers for Disease Control and Prevention worked with for over two decades. These men are well-versed on Schistosoma, and they decided to build a latrine.
“They knew the life cycle as well as we did, maybe better,” recalls Colley.
The car washers even made it an entrepreneurial pursuit by selling toilet paper for a shilling. However, when the rainy season came, the latrine was flooded, and all that waste was washed into the nearby lake where they worked daily.
This story illustrates just one of the challenges public health officials face in trying to incorporate wells and latrines into national programs to eliminate schistosomiasis. Studies like the one by Colley and his colleagues demonstrate the need to look beyond drug treatment when pushing to move from control to elimination of schistosomiasis.
“We can keep doing MDA, which is helpful, but eventually the worms may become drug resistant,” said Colley. “But with persistent hotspots now being found in everyone’s studies, we know we need to do better. That means we need to advocate for access to clean water sources, sanitation and perhaps ultimately vaccines, as well.”
Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.
Steven P. Maher, Amélie Vantaux, Victor Chaumeau, Adeline C. Y. Chua, Caitlin A. Cooper, Chiara Andolina, Julie Péneau, Mélanie Rouillier, Zaira Rizopoulos, Sivchheng Phal, Eakpor Piv, Chantrea Vong, Sreyvouch Phen, Chansophea Chhin, Baura Tat, Sivkeng Ouk, Bros Doeurk, Saorin Kim, Sangrawee Suriyakan, Praphan Kittiphanakun, Nana Akua Awuku, Amy J. Conway, Rays H. Y. Jiang, Bruce Russell, Pablo Bifani, Brice Campo, François Nosten, Benoît Witkowski & Dennis E. Kyle. Sci Rep 11, 19905 (2021). https://doi.org/10.1038/s41598-021-99152-9
Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca2+-dependent events. Little is known about TPCs in unicellular organisms despite their ancient origins. Here, we characterize a TPC from Toxoplasma gondii, the causative agent of toxoplasmosis. TgTPC is a member of a novel clad of TPCs in Apicomplexa, distinct from previously identified TPCs and only present in coccidians. We show that TgTPC localizes not to acidic organelles but to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. Conditional silencing of TgTPC resulted in progressive loss of apicoplast integrity, severely affecting growth and the lytic cycle. Isolation of TPC null mutants revealed a selective role for TPCs in replication independent of apicoplast loss that required conserved residues within the pore-lining region. Using a genetically-encoded Ca2+ indicator targeted to the apicoplast, we show that Ca2+ signals deriving from the ER but not from the extracellular space are selectively transmitted to the lumen. Deletion of the TgTPC gene caused reduced apicoplast Ca2+ uptake and membrane contact site formation between the apicoplast and the ER. Fundamental roles for TPCs in maintaining organelle integrity, inter-organelle communication and growth emerge.
Zhu-Hong Li, Thayer P King, Lawrence Ayong, Beejan Asady, Xinjiang Cai, Taufiq Rahman, Stephen A Vella, Isabelle Coppens, Sandip Patel, Silvia N J Moreno. Nat Commun. 2021 Oct 4;12(1):5802. doi: 10.1038/s41467-021-25987-5
Protein phosphorylation is involved in several key biological roles in the complex life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, and protein kinases are potential drug targets. Here, we report that the AGC essential kinase 1 (TcAEK1) exhibits a cytosolic localization and a higher level of expression in the replicative stages of the parasite. A CRISPR/Cas9 editing technique was used to generate ATP analog-sensitive TcAEK1 gatekeeper residue mutants that were selectively and acutely inhibited by bumped kinase inhibitors (BKIs). Analysis of a single allele deletion cell line (TcAEK1-SKO), and gatekeeper mutants upon treatment with inhibitor, showed that epimastigote forms exhibited a severe defect in cytokinesis. Moreover, we also demonstrated that TcAEK1 is essential for epimastigote proliferation, trypomastigote host cell invasion, and amastigote replication. We suggest that TcAEK1 is a pleiotropic player involved in cytokinesis regulation in T. cruzi and thus validate TcAEK1 as a drug target for further exploration. The gene editing strategy we applied to construct the ATP analog-sensitive enzyme could be appropriate for the study of other proteins of the T. cruzi kinome. IMPORTANCE Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are needed to combat this disease. In this work, we report the chemical and genetic validation of the protein kinase AEK1, which is essential for cytokinesis and infectivity, using a novel gene editing strategy.
Calcium ion (Ca2+) signaling is one of the most frequently employed mechanisms of signal transduction by eukaryotic cells, and starts with either Ca2+ release from intracellular stores or Ca2+ entry through the plasma membrane. In intracellular protist parasites Ca2+ signaling initiates a sequence of events that may facilitate their invasion of host cells, respond to environmental changes within the host, or regulate the function of their intracellular organelles. In this review we examine recent findings in Ca2+ signaling in two groups of intracellular protist parasites that have been studied in more detail, the apicomplexan and the trypanosomatid parasites.
Two UGA researchers are working to make it easier to develop effective treatments for malaria, a disease that sickens millions worldwide and kills hundreds of thousands each year.
In tropical climates around the globe, malaria poses a grave risk to already vulnerable populations. In 2019, the World Health Organization estimated that there were 229 million clinical cases of malaria worldwide and 409,000 deaths, usually in children below the age of five.
Currently, developing and testing drugs for malaria requires scientists to work in areas where the disease is prevalent or to work with expensive, hard-to-source equipment. Chester Joyner, an Assistant Professor in the Center for Vaccines and Immunology, and Dennis Kyle, Professor of Infectious Diseases and Cellular Biology, are working to reduce those barriers to malaria drug testing and development.
Joyner and Kyle aim to establish systems that rely on equipment most researchers can obtain: a petri dish. If successful, Joyner says this new culture system will reduce costs and be distributed more easily to advance drug and vaccine research. The University of Georgia College of Veterinary Medicine received a grant for malaria drug development and testing from the Bill & Melinda Gates Foundation.
Worldwide, there are many malaria-causing parasites that result in varying degrees of illness. Joyner and Kyle’s research focuses on defeating one of the most challenging: Plasmodium vivax. Unlike many other malaria parasites, P. vivax can lie dormant in the livers of its hosts—allowing the infected to travel abroad completely unaware that they’re carrying a potentially deadly passenger.
“Most infections with P. vivax are not due to new infections,” says Joyner. “These infections come from this parasite activating and potentially causing disease and sustaining transmission.”
Malaria disproportionately affects the poorest communities in the world, creating a cycle of disease and poverty that current treatments have improved but been unable to stop. However, treating the dormant forms of P. vivax has been particularly challenging because they can cause more harm than good in at-risk populations like pregnant women and people with certain blood conditions.
“We want researchers to have access to technologies to study P. vivax and develop new approaches to control and eliminate this parasite,” Joyner explains.
Background: Evidence indicates that whereas repeated rounds of mass drug administration (MDA) programs have reduced schistosomiasis prevalence to appreciable levels in some communities referred to here as responding villages (R). However, prevalence has remained high or less than anticipated in other areas referred to here as persistent hotspot villages (PHS). Using a cross-sectional quantitative approach, this study investigated the factors associated with sustained high Schistosoma mansoni prevalence in some villages despite repeated high annual treatment coverage in western Kenya.
Method: Water contact sites selected based on observation of points where people consistently go to collect water, wash clothes, bathe, swim or play (young children), wash cars and harvest sand were mapped using hand-held smart phones on the Commcare platform. Quantitative cross-sectional surveys on behavioral characteristics were conducted using interviewer-based semi-structured questionnaires administered to assess water usage/contact patterns and open defecation. Questionnaires were administered to 15 households per village, 50 pupils per school and 1 head teacher per school. One stool and urine sample was collected from 50 school children aged 9-12 year old and 50 adults from both responding (R) and persistent hotspot (PHS) villages. Stool was analyzed by the Kato-Katz method for eggs of S. mansoni and soil-transmitted helminths. Urine samples were tested using the point-of-care circulating cathodic antigen (POC-CCA) test for detection of S. mansoni antigen.
Results: There was higher latrine coverage in R (n = 6) relative to PHS villages (n = 6) with only 33% of schools in the PHS villages meeting the WHO threshold for boy: latrine coverage ratio versus 83.3% in R, while no villages met the girl: latrine ratio requirement. A higher proportion of individuals accessed unprotected water sources for both bathing and drinking (68.5% for children and 89% for adults) in PHS relative to R villages. In addition, frequency of accessing water sources was higher in PHS villages, with swimming being the most frequent activity. As expected based upon selection criteria, both prevalence and intensity of S. mansoni were higher in the PHS relative to R villages (prevalence: 43.7% vs 20.2%; P < 0.001; intensity: 73.8 ± 200.6 vs 22.2 ± 96.0, P < 0.0001), respectively.
Conclusion: Unprotected water sources and low latrine coverage are contributing factors to PHS for schistosomiasis in western Kenya. Efforts to increase provision of potable water and improvement in latrine infrastructure is recommended to augment control efforts in the PHS areas.
Musuva RM, Odiere MR, Mwinzi PNM, Omondi IO, Rawago FO, Matendechero SH, Kittur N, Campbell Jr CH, Colley DG. (2021) Unprotected water sources and low latrine coverage are contributing factors to persistent hotspots for schistosomiasis in western Kenya. PLoS ONE 16(9): e0253115. https://doi.org/10.1371/journal.pone.0253115
Though malaria was eliminated from the U.S. 70 years ago, the mosquito-borne disease caused by the Plasmodium parasite is still rampant in many parts of the world – nearly 40% of the world’s population is at risk of contracting it, and nearly 450,000 people die each year from it. With the rise of drug resistance, the current medical treatments aren’t enough to end this disease.
“Every drug treatment currently in use for malaria is showing resistance or reduced efficacy,” said Belen Cassera, a member of the University of Georgia’s Center for Tropical and Emerging Global Diseases. “Furthermore, there are very limited treatments for the most vulnerable – children and pregnant women. Over 60% of deaths are children under the age of 5.”
Cassera is co-leading the research team that recently received a $3.7 million grant from the National Institutes of Health to test two new drug candidates.
“These compounds are really promising as they are easy to synthesize, cheap, reliable, have a low toxicity profile, and kill the parasites fast,” said Cassera, associate professor in the Department of Biochemistry and Molecular Biology, part of the Franklin College of Arts and Sciences.
What’s unique about these compounds is that they can kill the parasite in three development stages in humans. Current treatments only target the blood stage, which is when clinical symptoms appear.
The life cycle of the Plasmodium parasite is complex. When an infected mosquito bites a person, just a small number of parasites – usually less than a hundred – are injected into the bite site and then travel to the liver, where they multiply in number to thousands. Once their numbers are sufficient enough, they invade the bloodstream and infect red blood cells.
When the number of parasites reaches 100 million, symptoms occur and some of the parasites develop into a sexual form, also known as the gametocyte stage. This is when symptoms occur. The sexual form is then transmitted back to the mosquito when the person is bitten again.
This complex life cycle makes it difficult to find a treatment that will eradicate the disease. Breaking the cycle of transmission between humans and mosquitos is key to accomplishing that goal. That’s why the team is excited about discovering compounds that can attack the parasite on multiple fronts.
“We are really a powerhouse team,” said Cassera. “We have a leading medicinal chemistry expert in Paul Carlier, the robust parasitology resources of UGA, and Max Totrov brings the machine-learning expertise to tie it all together.”
Cassera is a UGA Innovation Fellow, and she also credits the knowledge gained at UGA’s 2019 Innovation Bootcamp with helping her prepare a grant proposal that would be of particular interest to drug manufacturers.
“We started working with the Malaria Box from Medicines for Malaria Venture, and the discoveries we made in basic malaria biochemistry and medicinal chemistry really springboarded us to a new level and led us in this new direction,” Cassera said.
Cassera is leading the testing of the new chemical variations of the antimalarial compounds prepared by Carlier for effectiveness in cellular and animal models.
“My lab will be looking at levels of toxicity, the potential for resistance, and how well they work both directly on the parasite and in infected mice,” she said. “We’ll be performing the studies for making the go/no-go decision for these compounds.”
A joint patent application for both drug candidates was recently filed, and the team is optimistic that their research will yield fast-acting candidates for advanced pre-clinical evaluation.