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Cutaneous leishmaniasis (CL) is highly prevalent in rural and sylvatic regions of Latin America, with an estimated 55,000 annual cases. Diagnosis in resource-limited areas still relies on microscopy of dermal scrapings, while more sensitive methods like PCR are not attainable due to costs and lack of adequate health infrastructure. Isothermal amplification of Leishmania DNA can be performed without sophisticated equipment and training and may become a point of care (POC) test for health care centers with scarce resources. We evaluated the efficacy of recombinase-polymerase-amplification (RPA-LF) to diagnose CL in 226 patients attending a clinic in Puerto Maldonado within the Peruvian Amazon basin. Conventional PCR targeting kinetoplast DNA (kDNA-PCR) was used as the gold standard. Eight of 226 patients were considered true negatives (microscopy, kDNA-PCR, and RPA-LF negative), while RPA-LF resulted positive in 186 of 204 kDNA-PCR positive patients, yielding 91.2% (confidence interval [CI] = 86.5-94.4%) sensitivity and 93% (CI 88.6-95.8%) positive predictive value. There were 14% (32/226) discrepant samples alternating positive and negative results in similar proportions between both tests. Quantitative PCR used to resolve the discrepancies suggested that they occurred in samples with scarce parasite numbers as determined by high cycle threshold (Ct) values (≥32; cutoff 35.5). Microscopy had the lowest sensitivity of all methods (45.4%). Nested real-time PCR performed in 71 samples determined that Leishmania (Viannia) braziliensis was highly prevalent (69/71), and Leishmania (Viannia) lainsoni was present in only two isolates. Results indicated that RPA-LF has POC potential for CL endemic areas, yet further simplification and optimization coupled with field validation will be necessary to confirm its broad applicability.

Cryptosporidiosis is a major cause of diarrheal disease. The only drug approved for cryptosporidiosis has limited efficacy in high-risk populations. Therefore novel drugs are urgently needed. We have identified several enzymes as potential targets for drug development and we have optimized a rapid method to silence genes in Cryptosporidium. In this study, we knocked down expression of the four selected genes: Actin (Act), Apicomplexan DNA-binding protein (Ap2), Rhomboid protein 1 (Rom 1), and nucleoside diphosphate kinase (NDK). After gene silencing, we evaluated the role of each target on parasite development using in vitro models of excystation, invasion, proliferation, and egress. We showed that silencing of Act, Ap2, NDK, and Rom1 reduced invasion, proliferation, and egress of Cryptosporidium. However, silencing of NDK markedly inhibited Cryptosporidium proliferation (~70%). We used an infection model to evaluate the anticryptosporidial activity of ellagic acid (EA), an NDK inhibitor. We showed that EA (EC50 = 15–30 µM) reduced parasite burden without showing human cell toxicity. Here, we demonstrated the usefulness of a rapid silencing method to identify novel targets for drug development. Because EA is a dietary supplement already approved for human use, this compound should be studied as a potential treatment for cryptosporidiosis.

A gene silencing procedure on cysticerci of the taeniid cestode Taenia crassiceps is described. This is the first time this technique is reported in this species that is widely used as an animal model for human cysticercosis. Genome database searches were performed in order to find out if relevant genes involved in gene silencing and non-coding RNA processing, Argonaute and Dicer (AGO and Dcr) are present in T. crassiceps. We found three AGO and two Dcr orthologues that were designed TcAGO1, Tc2 and Tc3, as well as TcDcr1 and TcDcr2. In order to elucidate the evolutionary relationships of T. crassiceps TcAGO and TcDcr genes, separate phylogenetic analyses were carried out for each, including AGO and Dcr orthologues of other 20 platyhelminthes. Our findings showed a close phylogenetic relationship of TcAGO and TcDcr with those previously described for Echinococcus spp. Our RT-PCR studies demonstrated expression of all TcAGO and TcDcr orthologues. Our results show that the gene silencing machinery in T. crassiceps is functionally active by inducing silencing of TcEnoA (∼90%). These results clearly show that gene silencing using siRNAs can be used as a molecular methodology to study gene function in taeniid cestodes.

Introduction Cryptosporidium species are protozoan parasites that are important causes of diarrheal disease including waterborne outbreaks, childhood diarrhea in resource-poor countries, and diarrhea in compromised hosts worldwide. Recent studies highlight the importance of cryptosporidiosis in childhood diarrhea, malnutrition, and death in resource-poor countries. Despite this, only a single drug, nitazoxanide, has demonstrated efficacy in human cryptosporidiosis and its efficacy is limited in malnourished children and patients with HIV. Areas covered In this review, we highlight work on potential targets for chemotherapy and review progress on drug development. A number of new targets have been identified for chemotherapy and progress has been made at developing drugs for these targets. Targets include parasite kinases, nucleic acid synthesis and processing, proteases, and lipid metabolism. Other groups have performed high-throughput screening to identify potential drugs. Several compounds have advanced to large animal studies. Expert opinion Development of drugs for cryptosporidiosis has been plagued by a lack of success. Barriers have included poor correlations between in vitro activity and clinical success as well as frequent unanticipated adverse effects. Without a clear pathway forward, it is wise to maintain a diverse development pipeline. Drug developers should also realize that success will likely require a sustained, methodical effort.

Chagas disease is a lingering Public Health problem in Latin America with ∼5.7 million people infected with Trypanosoma cruzi. Transmission is still taking place in most countries of the Americas, including the United States. Dogs are frequently infected with T. cruzi and its high infection prevalence is associated with increased risk of Chagas disease in humans. The city of Mérida in the Yucatan peninsula is endemic for Chagas disease and canines are frequently infected with T. cruzi. The objective of this study was to evaluate the performance of a qualitative point of care (POC) molecular test (RPA-LF, recombinase polymerase amplification-lateral flow) developed in our laboratory for identifying infected dogs. We used retrospective samples of dogs that came for consultation because of cardiac alterations and proved to be infected with T. cruzi as determined by enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative PCR (qPCR). The analytical sensitivity indicated that RPA-LF amplified T. cruzi DNA in samples containing almost equal to one to two parasites per reaction. Serial twofold dilutions of T. cruzi epimastigotes showed that the test had 95% (19/20) repeatability at concentrations of two parasites per reaction. The test showed no cross reactivity with human DNA or other protozoan parasites (Trypanosoma rangeli, Leishmania spp., and Plasmodium spp.). RPA-LF had the capacity to amplify all discrete typing units (DTUs I-VI) of T. cruzi that circulate in domestic or extradomestic environments. The RPA-LF had 93.2% (95% confidence interval 87.2-98.1) sensitivity and excellent agreement with qPCR used as gold standard (Cohen's Kappa test = 0.963). ELISA was positive in 96.6% (85/88) of dogs, which together with the molecular tests confirmed the frequent contact with infected triatomine bugs in the city of Mérida. These preliminary results on the diagnostic efficacy of the RPA-LF deserve further large-scale field testing of this POC test for T. cruzi infection in endemic areas.

The parasites of the genus Cryptosporidium are important causes of diarrheal diseases, specifically cryptosporidiosis, worldwide. A major bottleneck for developing drugs and vaccines against cryptosporidiosis is the lack of methods to study gene function in this parasite. Silencing of genes by RNA interference (RNAi) is a powerful method to investigate gene function that has been widely used in the identification of targets for several pathogens. Unfortunately, as Cryptosporidium does not possess the enzymes of the RNAi pathway, its genes cannot be silenced by standard siRNA technology. To circumvent that problem, we have developed a novel strategy to knock down Cryptosporidium genes by reconstituting the effector arm of the siRNA pathway. We have induced silencing of several genes in Cryptosporidium by transfecting parasites with hybrid complexes formed between recombinant human Argonaute (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA). This novel methodology provides an effective strategy to study the role of selected genes in host–parasite interactions, and also can be used to identify potential targets for chemotherapy. The standardized methodology based on this strategy is described in this chapter.

Graphical abstract Silencing Cryptosporidium Protein kinase G blocks merozoite egress, Highlights • Cryptosporidium protein kinase G mRNA was silenced using siRNA, which led to decreased expression of the PKG protein. • After silencing, merozoite egress was blocked and merozoites retained within the host epithelical cells. • PKG plays an essential role in Cryptosporidium merozoite egress., Cryptosporidiosis is an obligate intracellular pathogen causing diarrhea. Merozoite egress is essential for infection to spread between host cells. However, the mechanisms of egress have yet to be defined. We hypothesized that Cyclic GMP-Dependent Protein Kinase G (PKG) may be involved in Cryptosporidium egress. In this study, Cryptosporidium parvum PKG was silenced by using antisense RNA sequences. PKG-silencing significantly inhibited egress of merozoites from infected HCT-8 cells into the supernatant and led to retention of intracellular forms within the host cells. This data identifies PKG as a key mediator of merozoite egress, a key step in the parasite lifecycle.

BackgroundCryptosporidiosis is a major cause of diarrheal disease. However, the only drug approved for cryptosporidiosis does not work well in high risk populations. Therefore, novel drugs are urgently needed. Then, the identification of novel is necessary to develop new therapies against this parasite. Recently, we have developed a rapid method to block gene expression in Cryptosporidium by using pre-assembled complexes of Cryptosporidium antisense RNA and human protein with slicer activity (Argonaute 2). We hypothesized that structural proteins, proteases, enzymes nucleotide synthesis and transcription factors are essential for parasite development, thus in this work we knock down expression of 4 selected genes: Actin, Apicomplexan DNA-binding protein (AP2), Rhomboid protein 1 (Rom 1) and nucleoside diphosphate kinase (NDK) and elucidated its role during invasion, proliferation and egress of Cryptosporidium.MethodsWe used protein transfection reagents (PTR) to introduce pre-assembled complexes of antisense RNA and human Argonaute 2 into Cryptosporidium parvum oocysts, the complexes blocked expression of Actin (Act), Transcription factor AP2 (AP2), nucleoside diphosphate kinase (DKN), and rhomboid protein 1 (Rom1). After gene silencing, we evaluated parasite reduction using In vitro models of excystation, invasion, proliferation and egress. We evaluated the potency of ellagic acid, a nucleoside diphosphate kinase inhibitor for anti-cryptosporidial activity using a model of in vitro infection with human HCT-8 cells.ResultsSilencing of Act, AP2, NDK and Rom1 reduce significantly invasion, proliferation and egress of Cryptosporidium. We showed that silencing of NDK markedly inhibited Cryptosporidium proliferation. This was confirmed by demonstration that ellagic acid reduced the number of parasites at micro molar concentrations (EC 50 =15-30 µM) without showing any toxic effect on human cells.ConclusionsOverall the results confirmed the usefulness RNA silencing can be used to identify novel targets for drug development against Cryptosporidium. We identified ellagic acid (EA), a nucleoside diphosphate kinase inhibitor also blocks Cryptosporidium proliferation. Since EA is a dietary supplement approved for human use, then this compound should be studied as a potential treatment for cryptosporidiosis.Author summaryThe World Health Organization reports diarrhea kills around 760,000 children under five every year. Cryptosporidium infection is a leading cause of diarrhea morbidity and mortality. Current therapies to treat this infection are suboptimal, therefore novel treatments are urgently needed. We used genetic tools to identify novel targets for drug development, thus in this work we evaluated the role of 4 genes during Cryptosporidium infection. We demonstrated that silencing of nucleoside-diphosphate kinase (NDK) drastically reduced invasion, proliferation and egress of this parasite. To validate these finding we used the Ellagic acid (EA) an inhibitor of NDK to treat infected intestinal cells. Our results confirmed that the EA blocks parasite proliferation on infected cells. Interestingly we observed that the ellagic acid also has anti cryptosporidial activity by inducing apoptosis. Since EA is a dietary supplement already approved for human use, then this compound has potential to be used as a rapid alternative to treat Cryptosporidiosis.

Despite the severity and global burden of Cryptosporidium infection, treatments are less than optimal, and there is no effective vaccine. Egress from host cells is a key process for the completion of the life cycle of apicomplexan parasites. For Plasmodium species, subtilisin-like serine protease (SUB1) is a key mediator of egress. For Toxoplasma species, calcium-dependent protein kinases (CDPKs) are critical. In this study, we characterized Cryptosporidium SUB1 expression and evaluated its effect using an infection model. We found increased expression between 12 and 20 h after in vitro infection, prior to egress. We induced silencing of SUB1 (ΔSUB1) mRNA using SUB1 single-stranded antisense RNA coupled with human Argonaute 2. Silencing of SUB1 mRNA expression did not affect parasite viability, excystation, or invasion of target cells. However, knockdown led to a 95% decrease in the proportion of released merozoites in vitro (P

Intestinal infection caused by Cryptosporidium is a major contributor to diarrhea morbidity and mortality in young children around the world. Current treatments for children suffering from cryptosporidiosis are suboptimal. Lactoferrin is a glycoprotein found in breast milk. It has showed bacteriostatic and antimicrobial activity in the intestine. However, the effects of lactoferrin on the intestinal parasite Cryptosporidium have not been reported. In this study, we investigated the anticryptosporidial activity of human lactoferrin on different stages of Cryptosporidium. Physiologic concentrations of lactoferrin killed Cryptosporidium parvum sporozoites, but had no significant effect on oocysts viability or parasite intracellular development. Since sporozoites are essential for the infection process, our data reinforce the importance of breastfeeding and point to the potential of lactoferrin as a novel therapeutic agent for cryptosporidiosis.

This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences.

Dogs are the principal reservoir hosts of zoonotic visceral leishmaniasis (VL) but current serological methods are not sensitive enough to detect all subclinically infected animals, which is crucial to VL control programs. Polymerase chain reaction (PCR) methods have greater sensitivity but require expensive equipment and trained personnel, impairing its implementation in endemic areas. We developed a diagnostic test that uses isothermal recombinase polymerase amplification (RPA) to detect Leishmania infantum. This method was coupled with lateral flow (LF) reading with the naked eye to be adapted as a point-of-care test. The L. infantum RPA-LF had an analytical sensitivity similar to real time-PCR, detecting DNA of 0.1 parasites spiked in dog blood, which was equivalent to 40 parasites/mL. There was no cross amplification with dog or human DNA or with Leishmania braziliensis, Leishmania amazonensis, or Trypanosoma cruzi. The test also amplified Leishmania donovani strains (N = 7). In a group of clinically normal dogs (N = 30), RPA-LF detected more subclinical infections than rK39 strip test, a standard serological method (50% versus 13.3% positivity, respectively; P = 0.005). Also, RPA-LF detected L. infantum in noninvasive mucosal samples of dogs with a sensitivity comparable to blood samples. This novel molecular test may have a positive impact in leishmaniasis control programs.

Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. Copyright © 2015 by The American Society of Tropical Medicine and Hygiene.

Cryptosporidiosis is increasingly recognized as an important global health concern. While initially reported in immunocompromised such as AIDS patients, cryptosporidiosis has now been documented as a major cause of childhood diarrhea and an important factor in childhood malnutrition. Currently, nitazoxanide is the only proven anti-parasitic treatment for Cryptosporidium infections. However, it is not effective in severely immunocompromised patients and there is limited data in infants. Immune reconstitution or decreased immunosuppression is critical to therapy in AIDS and transplant patients. This limitation of treatment options presents a major public health challenge given the important burden of disease. Repurposing of drugs developed for other indications and development of inhibitors for novel targets offer hope for improved therapies, but none have advanced to clinical studies.

Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Cryptosporidium hominis (C. hominis)-specific bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having μM antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy. 2009 Elsevier Ltd. All rights reserved.

This study describes the development and evaluation of an amplification-free molecular assay for detection of Cryptosporidium parvum oocysts. The assay employed a pair of oligonucleotide-functionalized gold nanoparticle (AuNP) probes that were complementary to adjacent sequences on C. parvum 18s rRNA. Hybridization of the probes to the target RNA resulted in the assembly of AuNPs into target-linked networks, which were detected both visibly and spectroscopically, by a redshift in the wavelength of light scattered by the gold nanoparticles. The limit of detection was between 4 × 10(5) and 4 × 10(6) copies of RNA per microliter reaction mix, when a short synthetic target or full-length in vitro transcribed target was employed. With total nucleic acids purified from C. parvum oocysts spiked into 100-mg stool, as few as 670 oocysts/μl reaction mix were detected. The ability to detect the nucleic acids of C. parvum oocysts in stool, without the need for complex amplification, offers unique advantages for such AuNP aggregation assays to be extended toward use in resource-limited settings where protozoan detection is needed most.

The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium . In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens.

Fasciola hepatica is the most widely distributed trematode infection in the world. Control efforts may be hindered by the lack of diagnostic capacity especially in remote endemic areas. Polymerase chain reaction (PCR)–based methods offer high sensitivity and specificity but require expensive technology. However, the recombinase polymerase amplification (RPA) is an efficient isothermal method that eliminates the need for a thermal cycler and has a high deployment potential to resource-limited settings. We report on the characterization of RPA and PCR tests to detect Fasciola infection in clinical stool samples with low egg burdens. The sensitivity of the RPA and PCR were 87% and 66%, respectively. Both tests were 100% specific showing no cross-reactivity with trematode, cestode, or nematode parasites. In addition, RPA and PCR were able to detect 47% and 26% of infections not detected by microscopy, respectively. The RPA adapted to a lateral flow platform was more sensitive than gel-based detection of the reaction products. In conclusion, the Fasciola RPA is a highly sensitive and specific test to diagnose chronic infection using stool samples. The Fasciola RPA lateral flow has the potential for deployment to endemic areas after further characterization.

Bumped kinase inhibitors (BKIs) of Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) are leading candidates for treatment of cryptosporidiosis-associated diarrhea. Potential cardiotoxicity related to anti-human ether-a-go-go potassium channel (hERG) activity of the first-generation anti-Cryptosporidium BKIs triggered further testing for efficacy. A luminescence assay adapted for high-throughput screening was used to measure inhibitory activities of BKIs against C. parvum in vitro. Furthermore, neonatal and interferon γ knockout mouse models of C. parvum infection identified BKIs with in vivo activity. Additional iterative experiments for optimum dosing and selecting BKIs with minimum levels of hERG activity and frequencies of other safety liabilities included those that investigated mammalian cell cytotoxicity, C. parvum proliferation inhibition in vitro, anti-human Src inhibition, hERG activity, in vivo pharmacokinetic data, and efficacy in other mouse models. Findings of this study suggest that fecal concentrations greater than parasite inhibitory concentrations correlate best with effective therapy in the mouse model of cryptosporidiosis, but a more refined model for efficacy is needed.

Cryptosporidium is recognized as one of the main causes of childhood diarrhea worldwide. However, the current treatment for cryptosporidiosis is suboptimal. Calcium flux is essential for entry in apicomplexan parasites. Calcium-dependent protein kinases (CDPKs) are distinct from protein kinases of mammals, and the CDPK1 of the apicomplexan Cryptosporidium lack side chains that typically block a hydrophobic pocket in protein kinases. We exploited this to develop bumped kinase inhibitors (BKIs) that selectively target CDPK1. We have shown that several BKIs of Cryptosporidium CDPK1 potently reduce enzymatic activity and decrease parasite numbers when tested in vitro. In the present work, we studied the anticryptosporidial activity of BKI-1517, a novel BKI. The half maximal effective concentration for Cryptosporidium parvum in HCT-8 cells was determined to be approximately 50 nM. Silencing experiments of CDPK1 suggest that BKI-1517 acts on CDPK1 as its primary target. In a mouse model of chronic infection, 5 of 6 SCID/beige mice (83.3%) were cured after treatment with a single daily dose of 120 mg/kg BKI-1517. No side effects were observed. These data support advancing BKI-1517 as a lead compound for drug development for cryptosporidiosis.

The protozoans Cryptosporidium parvum and Toxoplasma gondii are parasites of major health concern to humans. Both parasites contain a group of calcium-dependent protein kinases (CDPKs), which are found in plants and ciliates but not in humans or fungi. Here we describe a series of potent inhibitors that target CDPK1 in C. parvum (CpCDPK1) and T. gondii (TgCDPK1). These inhibitors are highly selective for CpCDPK1 and TgCDPK1 over the mammalian kinases SRC and ABL. Furthermore, they are able to block an early stage of C. parvum invasion of HCT-8 host cells, which is similar to their effects on T. gondii invasion of human fibroblasts.

Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, especially in immunocompromised individuals. CD4(+) T cells and interferon-gamma are key factors in the control of cryptosporidiosis in human and murine models. Previous studies led us to hypothesize that CD8(+) T cells contribute to clearance of intestinal epithelial Cryptosporidium infection in humans. We report here that antigen expanded sensitized CD8(+) T cells reduce the parasite load in infected intestinal epithelial cell cultures and lyse infected intestinal epithelial cells. These effects are most likely mediated by the release of cytotoxic granules. Elimination of parasites seems to require antigen presentation through both human leukocyte antigen (HLA)-A and HLA-B. These data suggest that cytotoxic CD8(+) T cells play a role in clearing Cryptosporidium from the intestine, a previously unrecognized feature of the human immune response against this parasite.

Cutaneous and mucosal leishmaniasis is widely distributed in Central and South America. Leishmania of the Viannia subgenus are the most frequent species infecting humans. L. (V.) braziliensis, L. (V.) panamensis are also responsible for metastatic mucosal leishmaniasis. Conventional or real time PCR is a more sensitive diagnostic test than microscopy, but the cost and requirement for infrastructure and trained personnel makes it impractical in most endemic regions. Primary health systems need a sensitive and specific point of care (POC) diagnostic tool. We developed a novel POC molecular diagnostic test for cutaneous leishmaniasis caused by Leishmania (Viannia) spp. Parasite DNA was amplified using isothermal Recombinase Polymerase Amplification (RPA) with primers and probes that targeted the kinetoplast DNA. The amplification product was detected by naked eye with a lateral flow (LF) immunochromatographic strip. The RPA-LF had an analytical sensitivity equivalent to 0.1 parasites per reaction. The test amplified the principal L. Viannia species from multiple countries: L. (V.) braziliensis (n = 33), L. (V.) guyanensis (n = 17), L. (V.) panamensis (n = 9). The less common L. (V.) lainsoni, L. (V.) shawi, and L. (V.) naiffi were also amplified. No amplification was observed in parasites of the L. (Leishmania) subgenus. In a small number of clinical samples (n = 13) we found 100% agreement between PCR and RPA-LF. The high analytical sensitivity and clinical validation indicate the test could improve the efficiency of diagnosis, especially in chronic lesions with submicroscopic parasite burdens. Field implementation of the RPA-LF test could contribute to management and control of cutaneous and mucosal leishmaniasis., Author Summary Cutaneous leishmaniasis is a parasitic disease transmitted by the bite of sandflies that produces skin ulcers. The severe, disfiguring form of the disease is characterized by parasite dissemination to the mucosa of the nose and palate. Current diagnosis is based on microscopy which has low sensitivity in chronic cases. Molecular methods (PCR) that detect parasite DNA are highly sensitive but costs and personnel training make it impossible to implement it in resource-limited areas. We developed a novel molecular method (RPA-LF) that could be applied in the field because it does not require sophisticated equipment. It is also very sensitive and specific to detect the principal Leishmania species that produce cutaneous leishmaniasis in Latin America. Future field implementation of RPA-LF could have a positive impact on disease management and control.

Fasciola hepatica is a zoonotic infection with a worldwide distribution. Autochthonous cases have not been reported in the Amazon region of Peru. Operculated eggs resembling F. hepatica were identified in the stools of five out of 215 subjects in a remote indigenous community of the Peruvian jungle. Polymerase chain reaction targeting Fasciola hepatica cytochrome oxidase subunit 1 (COI) gene and sequencing of the products confirmed Fasciola infection.

Cryptosporidiosis is a common cause of diarrhea morbidity and mortality worldwide. Research progress on this infection has been slowed by lack of methods to genetically manipulate Cryptosporidium parasites. Small interfering RNA (siRNA) is widely used to study gene function, but Cryptosporidium species lack the enzymes necessary to process siRNA. By preassembling complexes with the human enzyme Argonaute 2 (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA), we induced specific slicing in Cryptosporidium RNA targets. We demonstrated the reduction in expression of target genes at the mRNA and protein levels by transfecting live parasites with ssRNA-hAgo2 complexes. Furthermore we used this method to confirm the role of selected molecules during host cell invasion. This novel method provides a novel means of silencing Cryptosporidium genes to study their role in host-parasite interactions and as potential targets for chemotherapy.

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance.

Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS–DHFR specific inhibitors.

Diarrheal diseases cause more morbidity and mortality around the world than human immunodeficiency virus (HIV), malaria, or tuberculosis. Given that effective treatment of persistent diarrheal illness requires knowledge of the causative organism, diagnostic tests are of paramount importance. The protozoan parasites of the genus Cryptosporidium are increasingly recognized to be responsible for a significant portion of diarrhea morbidity. We present a novel nucleic acid test to detect the presence of Cryptosporidium species in DNA extracted from stool samples. The assay uses the isothermal amplification technique recombinase polymerase amplification (RPA) to amplify trace amounts of pathogen DNA extracted from stool to detectable levels in 30 min; products are then detected visually on simple lateral flow strips. The RPA-based Cryptosporidium assay (RPAC assay) was developed and optimized using DNA from human stool samples spiked with pathogen. It was then tested using DNA extracted from the stool of infected mice where it correctly identified the presence or absence of 27 out of 28 stool samples. It was finally tested using DNA extracted from the stool of infected patients where it correctly identified the presence or absence of 21 out of 21 stool samples. The assay was integrated into a foldable, paper and plastic device that enables DNA amplification with only the use of pipets, pipet tips, and a heater. The performance of the integrated assay is comparable to or better than polymerase chain reaction (PCR), without requiring the use of thermal cycling equipment. This platform can easily be adapted to detect DNA from multiple pathogens.

Cryptosporidiosis is an important cause of diarrhea worldwide. In normal hosts, infection is self-limited and associated with seroconversion and partial immunity to reinfection. Immunity is associated with interferon gamma (IFN) production. Cryptosporidium surface proteins gp15 and gp40 are among the immunodominant proteins in terms of antibody responses. We asked the question of whether these antigens also stimulate production of IFN in patients who have serologic evidence of prior infection. Whole blood from seropositive donors was stimulated with recombinant gp15 and gp 40 from Cryptosporidium hominis and Cryptosporidium parvum or His-tag controls. C. hominis gp15 stimulated increased production of IFN. By contrast, there was no significant increase after stimulation with C. parvum gp15 or either gp40 preparation. IFN production in response to C. hominis gp15 was noted in both CD4 + and CD8 + cells. This highlights the potential for C. hominis gp15 as a vaccine candidate for human cryptosporidiosis. Cryptosporidiosis is an important cause of diarrhea world- wide. 1 The host immune response controls the disease in im- munocompetent patients, and recovery is associated with re- sistance to reinfection. By contrast, cryptosporidiosis can be chronic and fatal in AIDS patients and malnourished chil- dren. Cytokines, particularly interferon gamma (IFN), are critical in the immune response controlling cryptosporidiosis. IFN-knockout mice develop chronic, severe infections. 2 Lymphocytes from people who have recovered from cryptosporidiosis produce IFN after antigen stimulation in vitro. 3,4 In volunteer studies, expression of IFN was associ- ated with resistance to infection and prior sensitization. 5 Thus, expression of IFN is an important marker of immunity to infection.

Febrile illnesses remain a major cause of morbidity and mortality in resource-poor countries, but too often, tests are not available to determine the causes, leading to misdiagnosis and inappropriate treatment. To determine the cause of febrile illnesses, we recovered the malaria smears from 102 children presenting with fever to Soddo Christian Hospital in Wolaitta Soddo, Ethiopia. DNA was isolated from the smears and evaluated by real-time polymerase chain reaction. We identified pathogen DNA with probes for Plasmodium spp., Streptococcus pneumoniae, Rickettsia spp., Salmonella spp., and Borrelia spp. Overall, we showed that it is possible to isolate high-quality DNA and identify treatable pathogens from malaria blood smears. Furthermore, our data showed that bacterial pathogens (especially Pneumococcus, Rickettsia spp., and Borrelia spp.) are common and frequently unrecognized but treatable causes of febrile illnesses in Ethiopian children.

Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50 nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues.

We report on a novel strategy for the detection of mRNA targets derived from Cryptosporidium parvum oocysts by the use of oligonucleotide-gold nanoparticles. Gold nanoparticles are functionalized with oligonucleotides which are complementary to unique sequences present on the heat shock protein 70 (HSP70) DNA/RNA target. The results indicate that the presence of HPS70 targets of increasing complexity causes the formation of oligonucleotide-gold nanoparticle networks which can be visually monitored via a simple colorimetric readout measured by a total internal reflection imaging setup. Furthermore, the induced expression of HSP70 mRNA in Cryptosporidium parvum oocysts via a simple heat shock process provides nonenzymatic amplification such that the HSP70 mRNA derived from as few as 5 × 10 3 purified C. parvum oocysts was successfully detected. Taken together, these results support the use of oligonucleotide-gold nanoparticles for the molecular diagnosis of cryptosporidiosis, offering new opportunities for the further development of point-of-care diagnostic assays with low-cost, robust reagents and simple colorimetric detection.

Cryptosporidium is an obligate intracellular protozoan parasite that is a major cause of diarrheal illness worldwide. Cryptosporidium primarily infects the distal small intestine. Immunocompetent hosts control and eliminate the infection, which typically causes acute, self-limited watery diarrhea lasting 5 to 10 days. However, in patients with defects in cellular immune responses (e.g., AIDS, malnutrition, or defects in the CD40-CD154 system), Cryptosporidium frequently causes persistent or chronic diarrhea and may also involve the biliary tract (40). In malnourished children, persistent diarrhea is associated with increased susceptibility to recurrent diarrheal episodes, which can lead to death or chronic nutritional and cognitive sequelae (1, 9, 33). Thus, the host immune response plays a critical role in the control of human cryptosporidiosis. Although extensive studies with various animal models have provided important insight into the host immune response towards Cryptosporidium parvum, the ability of these models to explain the human immune response is limited. The clinical picture in rodents differs from that in humans, as mice do not get diarrhea after infection. Nonhuman primates, although probably the best in vivo model to mimic human disease, are difficult to work with, expensive, and not widely available. Cryptosporidium hominis, the pathogen causing most human cryptosporidiosis, infects only humans and gnotobiotic pigs, thus limiting data from animal models. Most importantly, comparison of animal and human data has shown that the immune response towards Cryptosporidium in humans differs significantly from that in animals; for example, in mice gamma interferon (IFN-γ) production seems to be associated with the innate and primary immune responses (35, 47), whereas in humans it is most probably associated with the memory response towards the parasite (93). Conducting studies to elucidate human mucosal immune responses is difficult. Patients with a natural infection would be the ideal subjects to study, but it is difficult to identify cases. Healthy human volunteers can be studied, but they typically experience a milder illness than malnourished children and AIDS patients. Human intestinal tissue samples can be obtained only by invasive procedures, limiting the numbers of subjects and samples available. Some data can be obtained from in vitro infections, but most of the target cells are immortalized and may not be ideal for studying mechanisms involving apoptosis. Furthermore, the immune cells in the peripheral blood may exhibit properties different from the properties of cells found in the intestinal compartment. Thus, knowledge about the human immune response towards Cryptosporidium infection is far from complete. Still, important recent advances have been made. The goal of this paper is to review the current literature to provide an understanding of the human immune response towards the parasite. We include some relevant data from other models only when the data shed light on studies performed with human cells or tissues.

Cryptosporidiosis, caused by Cryptosporidium, causes self-limited diarrhea in normal hosts but may cause life-threatening diarrhea in immunocompromised persons. Cryptosporidium-induced manifestations, including weight-loss and intestinal physiological alterations are not noted in adult immunocompetent mice. So far, studies that have been used to test the therapeutic efficacy of drugs have been performed using various immunocompromised animal models. There is an urgent need of an immunocompetent small animal model that portrays Cryptosporidium-induced manifestations. In the current studies, we have compared two Cryptosporidium parvum pretreatment methods, we have hence used sodium hypochlorite or acidic water to treat Cryptosporidium parvum, followed by infection by oral gavage in adult immunocompetent C57BL6 mice. We demonstrated manifestations such as weight loss, intestinal structural and physiological alterations such as intestinal, villi blunting, and glucose malabsorption (as studied by the Ussing chamber technique) only in response to infection with C. parvum that has been treated with acidic water and not with sodium hypochlorite. These novel studies reveal that acidic water treatment of C. parvum results in manifestations of cryptosporidiosis in otherwise resistant immunocompetent mice. The current studies open up possibilities of using the normal immunocompetent mice model to test therapeutic drugs against cryptosporidiosis.