A new drug screening method was devised utilizing Trypanosoma cruzi cells that express the Escherichia coli ␤-galactosidase gene. Transfected parasites catalyze a colorimetric reaction with chlorophenol red ␤-D-galactopyranoside as substrate. Parasite growth in the presence of drugs in microtiter plates was quantitated with an enzyme-linked immunosorbent assay reader. The assay was performed with the mammalian form of T. cruzi that requires intracellular growth on a monolayer of fibroblast cells. To determine if selective toxicity to the parasites was occurring, the viability of the host cells in the drug was assayed with AlamarBlue. The drugs benznidazole, fluconazole, and amphotericin B were shown to inhibit the parasites at concentrations similar to those previously reported. Several compounds were tested that are inhibitors of glyceraldehyde-3-phosphate dehydrogenase of the related organisms Leishmania mexicana and Trypanosoma brucei. One of these compounds, 2-guanidino-benzimidazole, had an 50% inhibitory concentration of 10 M in our assay. Two derivatives of this compound were identified with in vitro activity at even lower concentrations. In addition, the assay was modified for testing compounds for lytic activity against the bloodstream form of the parasite under conditions used for storing blood products. Thus, an assay with ␤-galactosidase-expressing T. cruzi greatly simplifies screening drugs for selective anti-T. cruzi activity, and three promising new compounds have been identified.

Background Washington State served as the initial epicenter of the SARS-CoV-2 pandemic in the United States. An understanding of the risk factors and clinical outcomes of hospitalized patients with COVID-19 may provide guidance for management. Methods All laboratory-confirmed COVID-19 cases in adults admitted to an academic medical center in Seattle, WA between March 2 and March 26, 2020 were included. We evaluated individuals with and without severe disease, defined as admission to the intensive care unit or death. Results One-hundred-five COVID-19 patients were hospitalized. Thirty-five percent were admitted from a senior home or skilled nursing facility. The median age was 69 years and half were women. Three or more comorbidities were present in 55% of patients, with hypertension (59%), obesity (47%), cardiovascular disease (38%) and diabetes (33%) being the most prevalent. Most (63%) had symptoms for 5 days or longer prior to admission. Only 39% had fever in the first 24 hours, whereas 41% had hypoxia at admission. Seventy-three percent of patients had lymphopenia. Of 50 samples available for additional testing, no viral coinfections were identified. Severe disease occurred in 49%. Eighteen percent of patients were placed on mechanical ventilation and the overall mortality rate was 33%. Conclusions During the early days of the COVID-19 epidemic in Washington State, the disease had its greatest impact on elderly patients with medical comorbidities. We observed high rates of severe disease and mortality in our hospitalized patients.

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An estimated 12 million persons throughout the world suffer from the protozoan disease leishmaniasis. Current treatments have liabilities including poor activity against some forms of leishmaniasis, toxicity, or the need for parenteral administration. Higher throughput methods to screen chemical compounds are needed to facilitate the search for new antileishmania drugs. In the mammalian host, Leishmania parasites exist as amastigotes that replicate within macrophages. Therefore, an in vitro screening assay using intramacrophage amastigotes most closely represents the natural infection. We have transfected strains of Leishmania major and Leishmania amazonensis with the beta-lactamase gene, which catalyzes a colorimetric reaction with the substrate nitrocephin. The growth of these beta-lactamase-expressing Leishmania within macrophages was quantified in 96-well plates using an optical density plate reader, thus simplifying the methodology for scoring inhibitor assays. This simple and relatively inexpensive colorimetric assay helps improve throughput for screening compounds for antileishmania activity.

Trypanosoma cruzi is the protozoan parasite that causes Chagas’ disease, a frequently fatal illness affecting the heart and gastrointestinal systems. An estimated 16 million to 18 million people in Latin America and 50,000 to 100,000 people in the United States are infected with this pathogen. Treatment options for T. cruziinfections are suboptimal due to the toxicities and limited effectiveness of the available drugs. Azole antimicrobial agents have been discovered to have antitrypanosomal activity by inhibition of ergosterol synthesis. The triazole itraconazole was recently shown to produce a parasitologic cure rate of 53% in chronically infected patients (W. Apt et al., Am. J. Trop. Med. Hyg. 59:133–138, 1998), a result which may lead to more use of this family of drugs for the treatment of T. cruzi infections. In the experiments reported on here, resistance to azoles was induced in vitro by serial passage of mammalian-stage parasites in the presence of fluconazole for 4 months. These parasites were cross resistant to the other azoles, ketoconazole, miconazole, and itraconazole. They remained susceptible to benznidazole and amphotericin B. The azole-resistant phenotype was stable for more than 2 months of in vitro serial passage without fluconazole. In addition, the parasites resisted treatment in mice receiving ketoconazole. The rapid development of azole resistance inT. cruzi in vitro suggests that resistance to azole drugs has the potential to occur in patients and may pose an impediment to the progress being made in the treatment of T. cruziinfection.