Beyond schistosomiasis: unraveling co-infections and altered immunity.

Schistosomiasis is a neglected tropical disease caused by the helminth Schistosoma spp. and has the second highest global impact of all parasites. Schistosoma are transmitted through contact with contaminated fresh water predominantly in Africa, Asia, the Middle East, and South America. Due to the widespread prevalence of Schistosoma, co-infection with other infectious agents is common but often poorly described. Herein, we review recent literature describing the impact of Schistosoma co-infection between species and Schistosoma co-infection with blood-borne protozoa, soil-transmitted helminths, various intestinal protozoa, Mycobacterium, Salmonella, various urinary tract infection-causing agents, and viral pathogens. In each case, disease severity and, of particular interest, the immune landscape, are altered as a consequence of co-infection. Understanding the impact of schistosomiasis co-infections will be important when considering treatment strategies and vaccine development moving forward.

Canadian Blood Services traceback investigation of a suspected case of transfusion-transmitted malaria.

BACKGROUND: A 4-month-old infant hospitalized since birth received multiple blood transfusions. In March 2022, Plasmodium falciparum was confirmed with nucleic acid testing. As the mother was assessed as unlikely to be the source of infection, the blood operator initiated a traceback investigation for a potential blood donor source. The patient had received 13 red blood cell (RBC) transfusions (aliquoted from 11 donors), 4 apheresis platelet (PLT) transfusions and 16 buffy coat pooled PLT transfusions. The blood operator medical team developed a supplementary malaria infection risk questionnaire to identify donors at highest risk of life-time malaria infection, based on birthplace, residence, or travel in malaria-endemic regions. RESULTS: With 79 donors initially implicated, initial focus was on donors of RBC components. The 11 RBC donors were contacted and assessed using the supplementary questionnaire. Three donors, all of whom met current malaria-related donor eligibility criteria, were deemed high risk of prior malaria infection. These donors consented to P. falciparum serology and nucleic acid testing (NAT). One donor who was born and had resided in an endemic West African country for 14 years, was positive for P. falciparum by serology (indirect fluorescent antibody test) and NAT-(Ct ≥32). Lookback of this donor's transfused fresh co-components and prior donation identified no other malaria cases. CONCLUSION: This was a probable transfusion-transmitted malaria (TTM) case from an eligible donor who in retrospect was found to have unrecognized, asymptomatic, semi-immune malaria infection, and who was potentially infectious. Blood donor lack of recall of prior malaria infection does not negate the risk of TTM from those who have lived in malaria-endemic countries.

Aptamer-Based Technologies for Parasite Detection.

Centuries of scientific breakthroughs have brought us closer to understanding and managing the spread of parasitic diseases. Despite ongoing technological advancements in the detection, treatment, and control of parasitic illnesses, their effects on animal and human health remain a major concern worldwide. Aptamers are single-stranded oligonucleotides whose unique three-dimensional structures enable them to interact with high specificity and affinity to a wide range of targets. In recent decades, aptamers have emerged as attractive alternatives to antibodies as therapeutic and diagnostic agents. Due to their superior stability, reusability, and modifiability, aptamers have proven to be effective bioreceptors for the detection of toxins, contaminants, biomarkers, whole cells, pathogens, and others. As such, they have been integrated into a variety of electrochemical, fluorescence, and optical biosensors to effectively detect whole parasites and their proteins. This review offers a summary of the various types of parasite-specific aptamer-based biosensors, their general mechanisms and their performance.

Laboratory Features of Trichinellosis and Eosinophilia Threshold for Testing, Nunavik, Quebec, Canada, 2009-2019.

Prolonged eosinophilia is characteristic of trichinellosis. To determine the optimal eosinophil threshold for reflex Trichinella testing, we examined all 43 cases in Nunavik, Quebec, Canada, during 2009-2019. Using receiver operating characteristic analysis, we determined that eosinophil counts >0.8 × 109 cells/L should prompt consideration of trichinellosis and testing to rapidly identify potential outbreaks.

Specificity of SARS-CoV-2 Antibody Detection Assays against S and N Proteins among Pre-COVID-19 Sera from Patients with Protozoan and Helminth Parasitic Infections.

We aimed to assess the specificity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody detection assays among people with tissue-borne parasitic infections. We tested three SARS-CoV-2 antibody-detection assays (cPass SARS-CoV-2 neutralization antibody detection kit [cPass], Abbott SARS-CoV-2 IgG assay [Abbott Architect], and Standard Q COVID-19 IgM/IgG combo rapid diagnostic test [SD RDT IgM/SD RDT IgG]) among 559 pre-COVID-19 seropositive sera for several parasitic infections. The specificity of assays was 95 to 98% overall. However, lower specificity was observed among sera from patients with protozoan infections of the reticuloendothelial system, such as human African trypanosomiasis (Abbott Architect; 88% [95% CI, 75 to 95]) and visceral leishmaniasis (SD RDT IgG; 80% [95% CI, 30 to 99]), and from patients with recent malaria in areas of Senegal where malaria is holoendemic (ranging from 91% for Abbott Architect and SD RDT IgM to 98 to 99% for cPass and SD RDT IgG). For specimens from patients with evidence of past or present helminth infection overall, test specificity estimates were all ≥96%. Sera collected from patients clinically suspected of parasitic infections that tested negative for these infections yielded a specificity of 98 to 100%. The majority (>85%) of false-positive results were positive by only one assay. The specificity of SARS-CoV-2 serological assays among sera from patients with tissue-borne parasitic infections was below the threshold required for decisions about individual patient care. Specificity is markedly increased by the use of confirmatory testing with a second assay. Finally, the SD RDT IgG proved similarly specific to laboratory-based assays and provides an option in low-resource settings when detection of anti-SARS-CoV-2 IgG is indicated.

Babesia microti in a Canadian blood donor and lookback in a red blood cell recipient.

BACKGROUND AND OBJECTIVES: We describe the third documented case of autochthonous human babesiosis in Canada and the second in a Canadian blood donor. MATERIALS AND METHODS: Multiple laboratory investigations were carried out on the donor and the immunocompromised recipient of an associated, potentially infectious red blood cell product. RESULTS: The donor had not travelled except for outdoor exposure in south-eastern Manitoba, followed by illness and hospital admission. The donor had a notable parasitaemia, positive for Babesia microti using whole blood nucleic acid testing (NAT). The recipient was negative for B. microti by both serology and NAT. CONCLUSION: There was no evidence of transfusion-transmitted babesiosis.

First Human Case of Metacestode Infection Caused by Versteria sp. in a Kidney Transplant Recipient.

Cestodes are emerging agents of severe opportunistic infections among immunocompromised patients. We describe the first case of human infection, with the recently-proposed genus Versteria causing an invasive, tumor-like hepatic infection with regional and distant extension in a 53-year-old female kidney transplant recipient from Atlantic Canada.

Cutaneous leishmaniasis in the 21st century: from the laboratory to the bedside.

PURPOSE OF REVIEW: Despite modern advances in molecular diagnostic tools and a better understanding of its complex pathophysiology, cutaneous leishmaniasis, a neglected tropical disease, remains a major global health problem. Laboratory methods to inform prognosis and treatment are not widely available, the therapeutic options are limited and have significant adverse effects, and emergence of drug resistance is a further complication. New advances in the understanding of the role of Leishmania RNA virus (LRV) as a prognostic factor, speciation methods and antimicrobial resistance testing and their limitations will be discussed. RECENT FINDINGS: LRV, an intracytoplasmic endosymbiont found mostly in Leishmania spp. associated with more severe disease, appears to play a role in modulating the host immune response and has been associated with treatment failure in some Viannia subgenus species. Proper speciation is an important guide to management. However, recent findings have demonstrated significant heterogeneity of results related to differences in genotyping methods. SUMMARY: Recognition of the role of LRV in immune modulation and response to treatment along with more accessible tools for its detection to guide management at the bedside should allow a better individualized approach. Improving accessibility and standardization of speciation methods and antimicrobial susceptibility testing should be major goals to improve cutaneous leishmaniasis management in the 21st century.

Peptidyl-prolyl cis-trans isomerase A - A novel biomarker of multi-episodic (recurrent) ocular toxoplasmosis.

Ocular toxoplasmosis (OT) is the most common etiology of posterior uveitis. The high incidence of macular scarring associated with OT is a leading cause of visual morbidity. Serum biomarkers of the disease would aid in its diagnosis. This study sought, for the first time, to elucidate serum biomarkers for OT by mass spectrometry. Blood samples were collected from four groups of nine patients each; toxoplasmosis IgG-with no history of uveitis, non-toxoplasmosis uveitis, first episode OT, and symptomatic recurrent OT. Serum was isolated and subjected to proteomics analysis using 2-dimensional gel electrophoresis (2D-GE) and surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS). Selected proteins were further separated by SDS-PAGE and sequenced using tandem MS. Results were cross-validated with a T. gondii outbreak biomarker database that occurred in Brazil. Fifty markers of OT and 46 markers of recurrent disease were discovered by SELDI-MS of which 30 and 15, respectively, were cross-validated. 2D-GE analysis yielded 57 bands, selected based on the intensity of the bands, leading to the identification of 20 proteins. Eleven of those identified candidates were also found by SELDI-MS. Four candidates were chosen for immunoblotting. One serum protein, peptidyl-prolyl cis-trans isomerase A (PPIA), was confirmed as a biomarker of multi-episodic OT by immunoblotting in patients. PPIA can identify the patient with active recurrent OT from acute OT, other forms of uveitis and other parasitic infections. A validated PPIA assay may have a role in the diagnosis of the atypical OT patient before more invasive anterior chamber or vitreous tap is performed for PCR analysis or for Goldmann-Witner coefficient calculations. Base-line PPIA levels need to be studied to understand its possible use when deciding for prophylactic antibiotic use in the immunosuppressed sero-positive patient.

Development of a sandwich ELISA to detect Leishmania 40S ribosomal protein S12 antigen from blood samples of visceral leishmaniasis patients.

BACKGROUND: Visceral leishmaniasis (VL), caused by Leishmania donovani complex parasites, is a neglected parasitic disease that is generally fatal if untreated. Despite decades of research to develop a sensitive VL diagnostic test, definitive diagnosis of VL still mainly relies on the visualization of the parasite in aspirates from the spleen, liver or bone marrow, an invasive and dangerous process with variable sensitivity. A sensitive assay that can detect Leishmania antigen from blood samples will help confirm cause, cure or recurrence of VL. METHODS: In this study, rabbit polyclonal antibodies were raised against eight recombinant Leishmania proteins that are highly abundant in Leishmania. The antibodies were purified and labeled with biotin for developing a prototype sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The ELISA for the Leishmania 40S ribosomal protein S12 detected target antigen with the highest sensitivity and specificity and could detect 1 pg of purified protein or as few as 60 L. donovani parasites. The 40S ribosomal protein S12 sandwich ELISA could detect the target antigen from Peripheral Blood Mononuclear Cell (PBMC) samples in 68% of VL patients and post-kala-azar dermal leishmaniasis (PKDL) patients, providing an estimation of parasitemia ranging from 15 to 80 amastigotes per ml of blood. CONCLUSION: These results indicate that the 40S ribosomal protein S12 sandwich ELISA warrants further tests with more clinical samples of VL patients and other parasitic diseases. It is hopeful that this ELISA could become a useful tool for confirming VL diagnosis, monitoring treatment progress, disease recurrence and possibly detecting asymptomatic Leishmania infections with a high parasite load.

Characterization and Diagnostic Application of Trypanosoma cruzi Trypomastigote Excreted-Secreted Antigens Shed in Extracellular Vesicles Released from Infected Mammalian Cells.

Chagas disease, caused by Trypanosoma cruzi, although endemic in many parts of Central and South America, is emerging as a global health threat through the potential contamination of blood supplies. Consequently, in the absence of a gold standard assay for the diagnosis of Chagas disease, additional antigens or strategies are needed. A proteomic analysis of the trypomastigote excreted-secreted antigens (TESA) associated with exosomal vesicles shed by T. cruzi identified ∼80 parasite proteins, with the majority being trans-sialidases. Mass spectrometry analysis of immunoprecipitation products performed using Chagas immune sera showed a marked enrichment in a subset of TESA proteins. Of particular relevance for diagnostic applications were the retrotransposon hot spot (RHS) proteins, which are absent in Leishmania spp., parasites that often confound diagnosis of Chagas disease. Interestingly, serological screens using recombinant RHS showed a robust immunoreactivity with sera from patients with clinical stages of Chagas ranging from asymptomatic to advance cardiomyopathy and this immunoreactivity was comparable to that of crude TESA. More importantly, no cross-reactivity with RHS was detected with sera from patients with malaria, leishmaniasis, toxoplasmosis, or African sleeping sickness, making this protein an attractive reagent for diagnosis of Chagas disease.

A vaccine consisting of Schistosoma mansoni cathepsin B formulated in Montanide ISA 720 VG induces high level protection against murine schistosomiasis.

BACKGROUND: Schistosomiasis is the most important human helminth infection due to its impact on public health. The clinical manifestations are chronic and significantly decrease an individual's quality of life. Infected individuals suffer from long-term organ pathologies including fibrosis which eventually leads to organ failure. The development of a vaccine against this parasitic disease would contribute to a long-lasting decrease in disease spectrum and transmission. METHOD: Our group has chosen Schistosoma mansoni (Sm) cathepsin B, a peptidase involved in parasite feeding, as a prospective vaccine candidate. Our experimental formulation consisted of recombinant Sm-cathepsin B formulated in Montanide ISA 720 VG, a squalene based adjuvant containing a mannide mono-oleate emulsifier. Parasitological burden was assessed by determining adult worm, hepatic egg, and intestinal egg numbers in each mouse. Serum was used in ELISAs to evaluate production of antigen-specific antibodies, and isolated splenocytes were stimulated with the antigen for the analysis of cytokine secretion levels. RESULTS: The Sm-cathepsin B and Montanide formulation conferred protection against a challenge infection by significantly reducing all forms of parasitological burdens. Worm burden, hepatic egg burden and intestinal egg burden were decreased by 60%, 6%, and 56%, respectively in immunized animals compared to controls (P = 0.0002, P < 0.0001, P = 0.0009, respectively). Immunizations with the vaccine elicited robust production of Sm-cathepsin B specific antibodies (endpoint titers = 122,880). Both antigen-specific IgG1 and IgG2c titers were observed, with the former having more elevated titers. Furthermore, splenocytes isolated from the immunized animals, compared to control animals, secreted higher levels of key Th1 cytokines, IFN-γ, IL-12, and TNF-α, as well as the Th2 cytokines IL-5 and IL-4 when stimulated with recombinant Sm-cathepsin B. The Th17 cytokine IL-17, the chemokine CCL5, and the growth factor GM-CSF were also significantly increased in the immunized animals compared to the controls. CONCLUSION: The formulation tested in this study was able to significantly reduce all forms of parasite burden, stimulate robust production of antigen-specific antibodies, and induce a mixed Th1/Th2 response. These results highlight the potential of Sm-cathepsin B/Montanide ISA 720 VG as a vaccine candidate against schistosomiasis.

Apolipoprotein A-I truncations in Chagas disease are caused by cruzipain, the major cysteine protease of Trypanosoma cruzi.

Trypanosoma cruzi is the etiologic agent of Chagas disease. Approximately 10 million people are infected worldwide. We have previously reported that in individuals infected with T. cruzi, apolipoprotein A-I (Apo A-I), the major structural component of host high-density lipoprotein, was truncated into fragments that are specific to Chagas disease and have the potential to be used as diagnostic biomarkers. We investigated the possibility that cruzipain, the major cysteine protease of T. cruzi, is responsible for truncating host Apo A-I. We found that due to Apo A-I truncation, the high-density lipoprotein subspecies profile is altered in individuals with Chagas disease compared with healthy controls. Western blot analysis revealed that both purified Apo A-I protein and Apo A-I in the high-density lipoprotein complex were susceptible to cruzipain cleavage, and the sizes of the truncation product in the latter matched the sizes of Apo A-I biomarkers. We also found that in vitro feeding T. cruzi-infected differentiated human adipocytes with purified human high-density lipoprotein led to the appearance of the biomarker fragments of Apo A-I. Cruzipain is found both on the cytoplasmic membrane and in the internal lysosomal structure of T. cruzi. We demonstrate that cruzipain from both sources contributes to the production of Apo A-I truncation in the biomarker set.

A family cluster of Chagas disease detected through selective screening of blood donors: A case report and brief review.

Chagas disease (CD) is a protozoan infection caused by Trypanosoma cruzi, which is transmitted by triatomine insect vectors in parts of Latin America. In a nonendemic country, such as Canada, spread can still occur via vertical transmission, and infected blood or organ donations. The Canadian Blood Services and Héma-Québec have both implemented selective screening of blood donors for CD based on risk factors. In 2011, Héma-Québec identified two seropositive 'at-risk' Chilean siblings who had donated blood in Montreal, Quebec. They were referred to the JD MacLean Centre for Tropical Diseases (Montreal, Quebec) for confirmatory testing (T cruzi excreted-secreted antigen ELISA, polymerase chain reaction and/or radioimmunoprecipitation assay) and follow-up. Screening of the rest of the family revealed two other seropositive family members (the mother and sister). While their geographical history in Chile suggests vectorial transmission, this family cluster of CD raises the possibility of vertical transmission. Congenital infection should always be considered among CD-positive mothers and pregnant women. With blood donor screening, Canadian physicians will increasingly see patients with CD and should know how to manage them appropriately. In addition to the case presentation, the authors review the transmission, screening and clinical management of CD in a nonendemic context.

La maladie de Chagas (MC) est une infection protozoaire causée par le Trypanosoma cruzi, transmis par des vecteurs d'insectes du genre triatomine dans certaines régions d'Amérique latine. Dans un pays non endémique comme le Canada, la propagation est possible par transmission verticale et par les dons de sang ou d'organes infectés. La Société canadienne du sang et Héma-Québec ont tous deux adopté le dépistage sélectif des donneurs de sang pour déceler la MC en fonction des facteurs de risque. En 2011, Héma-Québec a repéré deux membres d'une fratrie chilienne séropositifs « à risque ¼ qui avaient donné du sang à Montréal, au Québec. Ils ont été orientés vers le Centre des maladies tropicales JD MacLean de Montréal pour subir des tests de confirmation (test ELISA des antigènes excrétés-sécrétés par T cruzi, réaction en chaîne de la polymérase ou test de radio-immunoprécipitation) et profiter d'un suivi. Le dépistage du reste de la famille a révélé deux autres membres séropositifs (la mère et la sœur). Étant donné leur origine géographique, on pourrait subodorer une transmission vectorielle, mais la grappe familiale de MC soulève la possibilité d'une transmission verticale. L'infection congénitale devrait toujours être envisagée chez les mères et les femmes enceintes positives à la MC. Grâce au dépistage des donneurs de sang, les médecins canadiens verront de plus en plus de patients atteints de la MC. Ils devraient donc savoir comment la prendre en charge correctement. En plus de la présentation de cas, les auteurs passent en revue la transmission, le dépistage et la prise en charge clinique de la MC dans un contexte non endémique.

Reversible cysteine protease inhibitors show promise for a Chagas disease cure.

The cysteine protease cruzipain is essential for the viability, infectivity, and virulence of Trypanosoma cruzi, the causative agent of Chagas disease. Thus, inhibitors of cruzipain are considered promising anti-T. cruzi chemotherapeutic agents. Reversible cruzipain inhibitors containing a nitrile "warhead" were prepared and demonstrated 50% inhibitory concentrations (IC50s) as potent as 1 nM in baculovirus-generated cruzipain enzyme assays. In epimastigote and intracellular amastigote in vitro assays, the most potent compounds demonstrated antiparasitic behavior in the 5 to 10 µM IC50 range; however, trypomastigote production from the amastigote form was ∼90 to 95% inhibited at 2 µM. Two key compounds, Cz007 and Cz008, with IC50s of 1.1 and 1.8 nM, respectively, against the recombinant enzyme were tested in a murine model of acute T. cruzi infection, with oral dosing in chow for 28 days at doses from 3 to 50 mg/kg of body weight. At 3 mg/kg of Cz007 and 3 mg/kg of Cz008, the blood parasitemia areas under the concentration-time curves were 16% and 25% of the untreated group, respectively. At sacrifice, 24 days after immunosuppression with cyclophosphamide, parasite presence in blood, heart, and esophagus was evaluated. Based on negative quantitative PCR results in all three tissues, cure rates in surviving animals were 90% for Cz007 at 3 mg/kg, 78% for Cz008 at 3 mg/kg, and 71% for benznidazole, the control compound, at 50 mg/kg.

Evaluation of selective screening of donors for antibody to Trypanosoma cruzi: seroprevalence of donors who answer "no" to risk questions.

BACKGROUND: Selective testing of donors for Trypanosoma cruzi infection relies on identification of at-risk donors with screening questions. Using risk modeling and a seroprevalence study, we evaluated the risk of questions failing to identify T. cruzi antibody-positive donors. STUDY DESIGN AND METHODS: The rate of donors with unreported risk was estimated by a telephone survey of 2677 donors who answered "no" to risk questions. The number of T. cruzi antibody-positive donors missed by risk questions was estimated from the product of this rate and the selective testing T. cruzi antibody-positive rate. The 95% confidence interval (CI) was estimated by Monte Carlo simulation. To test the model, 60,132 donors were tested for T. cruzi antibody (26% of donors in selected regions, Phase I). In Winnipeg, Manitoba, the highest-risk region, 26,915 donors were tested (92.5% of donors, Phase II). RESULTS: In the telephone survey, 21 (0.8%) donors reported risk factors that would have identified them for selective testing. Seven were born in Mexico or Central or South America, five had travel risk, and nine had mother or maternal grandmother risk. The 95% CI for predicted number of T. cruzi antibody-positive donors answering "no" to risk questions was 0.71 to 4.38. In Phase I, one Winnipeg donor confirmed positive but had answered risk questions correctly. No other positive donations were identified. CONCLUSION: The estimated risk of T. cruzi-positive donors who answer "no" to risk questions is low and is confirmed by the seroprevalence among these donors.

Serum biomarkers predictive of cure in Chagas disease patients after nifurtimox treatment.

BACKGROUND: Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, remains an important public health issue in many Central and South American countries, as well as non-endemic areas with high rates of immigration from these countries. Existing treatment options for CD are limited and often unsatisfactory. Moreover the lack of post-treatment tests of cure limits the development of new drugs. To address this issue, we sought to identify serum biomarkers following nifurtimox (Nfx) treatment that could be used as an early test of cure and/or markers of a therapeutic response. METHODS: Human sera from Chagas patients pre- and post-treatment with Nfx (n = 37) were compared to samples from healthy subjects (n = 37) using a range of proteomic and immunologic techniques. Biomarker peaks with the best discriminatory power were further characterized. RESULTS: Using serum samples (n = 111), we validated the presence of five key biomarkers identified in our previous study, namely human apolipoprotein A-I (APOA1) and specific fragments thereof and one fragment of human fibronectin (FN1). In chagasic serum samples all biomarkers except full-length APOA1 were upregulated. These five biomarkers returned to normal in 43% (16/37) of the patients treated with Nfx at three years after treatment. CONCLUSIONS: The normalization of biomarker patterns strongly associated with CD suggests that these markers can be used to identify patients in whom Nfx treatment is successful. We believe that these are the first biomarkers predictive of cure in CD patients.

Trypanosoma cruzi infection and host lipid metabolism.

Trypanosoma cruzi is the causative agent of Chagas disease. Approximately 8 million people are thought to be affected worldwide. Several players in host lipid metabolism have been implicated in T. cruzi-host interactions in recent research, including macrophages, adipocytes, low density lipoprotein (LDL), low density lipoprotein receptor (LDLR), and high density lipoprotein (HDL). All of these factors are required to maintain host lipid homeostasis and are intricately connected via several metabolic pathways. We reviewed the interaction of T. cruzi with each of the relevant host components, in order to further understand the roles of host lipid metabolism in T. cruzi infection. This review sheds light on the potential impact of T. cruzi infection on the status of host lipid homeostasis.

A cysteine protease inhibitor rescues mice from a lethal Cryptosporidium parvum infection.

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitor N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits C. parvum growth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-γR-KO) mouse model, which is highly susceptible to C. parvum, oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in the C. parvum genome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicity in vitro and in vivo, and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis.