A fractional perspective on the transmission dynamics of a parasitic infection, considering the impact of both strong and weak immunity.

Infectious disease cryptosporidiosis is caused by the cryptosporidium parasite, a type of parasitic organism. It is spread through the ingestion of contaminated water, food, or fecal matter from infected animals or humans. The control becomes difficult because the parasite may remain in the environment for a long period. In this work, we constructed an epidemic model for the infection of cryptosporidiosis in a fractional framework with strong and weak immunity concepts. In our analysis, we utilize the well-known next-generation matrix technique to evaluate the reproduction number of the recommended model, indicated by [Formula: see text]. As [Formula: see text], our results show that the disease-free steady-state is locally asymptotically stable; in other cases, it becomes unstable. Our emphasis is on the dynamical behavior and the qualitative analysis of cryptosporidiosis. Moreover, the fixed point theorem of Schaefer and Banach has been utilized to investigate the existence and uniqueness of the solution. We identify suitable conditions for the Ulam-Hyers stability of the proposed model of the parasitic infection. The impact of the determinants on the sickness caused by cryptosporidiosis is highlighted by the examination of the solution pathways using a novel numerical technique. Numerical investigation is conducted on the solution pathways of the system while varying various input factors. Policymakers and health officials are informed of the crucial factors pertaining to the infection system to aid in its control.

Commensal Cryptosporidium colonization elicits a cDC1-dependent Th1 response that promotes intestinal homeostasis and limits other infections.

Cryptosporidium can cause severe diarrhea and morbidity, but many infections are asymptomatic. Here, we studied the immune response to a commensal strain of Cryptosporidium tyzzeri (Ct-STL) serendipitously discovered when conventional type 1 dendritic cell (cDC1)-deficient mice developed cryptosporidiosis. Ct-STL was vertically transmitted without negative health effects in wild-type mice. Yet, Ct-STL provoked profound changes in the intestinal immune system, including induction of an IFN-γ-producing Th1 response. TCR sequencing coupled with in vitro and in vivo analysis of common Th1 TCRs revealed that Ct-STL elicited a dominant antigen-specific Th1 response. In contrast, deficiency in cDC1s skewed the Ct-STL CD4 T cell response toward Th17 and regulatory T cells. Although Ct-STL predominantly colonized the small intestine, colon Th1 responses were enhanced and associated with protection against Citrobacter rodentium infection and exacerbation of dextran sodium sulfate and anti-IL10R-triggered colitis. Thus, Ct-STL represents a commensal pathobiont that elicits Th1-mediated intestinal homeostasis that may reflect asymptomatic human Cryptosporidium infection.

Genomic Spectrum and Phenotypic Heterogeneity of Human IL-21 Receptor Deficiency.

Biallelic inactivating mutations in IL21R causes a combined immunodeficiency that is often complicated by cryptosporidium infections. While eight IL-21R-deficient patients have been reported previously, the natural course, immune characteristics of disease, and response to hematopoietic stem cell transplantation (HSCT) remain to be comprehensively examined. In our study, we have collected clinical histories of 13 patients with IL-21R deficiency from eight families across seven centers worldwide, including five novel patients identified by exome or NGS panel sequencing. Eight unique mutations in IL21R were identified in these patients, including two novel mutations. Median age at disease onset was 2.5 years (0.5-7 years). The main clinical manifestations were recurrent bacterial (84.6%), fungal (46.2%), and viral (38.5%) infections; cryptosporidiosis-associated cholangitis (46.2%); and asthma (23.1%). Inflammatory skin diseases (15.3%) and recurrent anaphylaxis (7.9%) constitute novel phenotypes of this combined immunodeficiency. Most patients exhibited hypogammaglobulinemia and reduced proportions of memory B cells, circulating T follicular helper cells, MAIT cells and terminally differentiated NK cells. However, IgE levels were elevated in 50% of IL-21R-deficient patients. Overall survival following HSCT (6 patients, mean follow-up 1.8 year) was 33.3%, with pre-existing organ damage constituting a negative prognostic factor. Mortality of non-transplanted patients (n = 7) was 57.1%. Our detailed analysis of the largest cohort of IL-21R-deficient patients to date provides in-depth clinical, immunological and immunophenotypic features of these patients, thereby establishing critical non-redundant functions of IL-21/IL-21R signaling in lymphocyte differentiation, humoral immunity and host defense against infection, and mechanisms of disease pathogenesis due to IL-21R deficiency. Outcome following HSCT depends on prior chronic infections and organ damage, which should thus be considered as early as possible following molecular diagnosis.

Use-case scenarios for an anti-Cryptosporidium therapeutic.

Cryptosporidium is a widely distributed enteric parasite that has an increasingly appreciated pathogenic role, particularly in pediatric diarrhea. While cryptosporidiosis has likely affected humanity for millennia, its recent "emergence" is largely the result of discoveries made through major epidemiologic studies in the past decade. There is no vaccine, and the only approved medicine, nitazoxanide, has been shown to have efficacy limitations in several patient groups known to be at elevated risk of disease. In order to help frontline health workers, policymakers, and other stakeholders translate our current understanding of cryptosporidiosis into actionable guidance to address the disease, we sought to assess salient issues relating to clinical management of cryptosporidiosis drawing from a review of the literature and our own field-based practice. This exercise is meant to help inform health system strategies for improving access to current treatments, to highlight recent achievements and outstanding knowledge and clinical practice gaps, and to help guide research activities for new anti-Cryptosporidium therapies.

A host cell long noncoding RNA NR_033736 regulates type I interferon-mediated gene transcription and modulates intestinal epithelial anti-Cryptosporidium defense.

The gastrointestinal epithelium guides the immune system to differentiate between commensal and pathogenic microbiota, which relies on intimate links with the type I IFN signal pathway. Epithelial cells along the epithelium provide the front line of host defense against pathogen infection in the gastrointestinal tract. Increasing evidence supports the regulatory potential of long noncoding RNAs (lncRNAs) in immune defense but their role in regulating intestinal epithelial antimicrobial responses is still unclear. Cryptosporidium, a protozoan parasite that infects intestinal epithelial cells, is an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children in developing countries. Recent advances in Cryptosporidium research have revealed a strong type I IFN response in infected intestinal epithelial cells. We previously identified a panel of host cell lncRNAs that are upregulated in murine intestinal epithelial cells following microbial challenge. One of these lncRNAs, NR_033736, is upregulated in intestinal epithelial cells following Cryptosporidium infection and displays a significant suppressive effect on type I IFN-controlled gene transcription in infected host cells. NR_033736 can be assembled into the ISGF3 complex and suppresses type I IFN-mediated gene transcription. Interestingly, upregulation of NR_033736 itself is triggered by the type I IFN signaling. Moreover, NR_033736 modulates epithelial anti-Cryptosporidium defense. Our data suggest that upregulation of NR_033736 provides negative feedback regulation of type I IFN signaling through suppression of type I IFN-controlled gene transcription, and consequently, contributing to fine-tuning of epithelial innate defense against microbial infection.

Delayed Time to Cryptosporidiosis in Bangladeshi Children is Associated with Greater Fecal IgA against Two Sporozoite-Expressed Antigens.

Cryptosporidiosis is common in early childhood, and both diarrheal and subclinical infections are associated with adverse developmental outcomes. Improved therapeutic medications may help reduce the burden of cryptosporidial diarrhea; however, an effective vaccine would be better able to prevent the detrimental impact of both diarrheal and subclinical disease. A more complete understanding of naturally occurring immunity may further inform strategies to develop an effective vaccine. In this prospective cohort study of Bangladeshi children, greater fecal IgA at 12 months, but not plasma IgG, directed against two sporozoite-expressed, immunodominant and vaccine candidate antigens was associated with delayed time to subsequent cryptosporidiosis to 3 years of life. These findings extend prior work and further support the role of mucosal antibody responses in naturally developing protective immunity to Cryptosporidium.

Development of an immunocompetent mouse model susceptible to Cryptosporidium tyzzeri infection.

AIMS: Immunocompromised mice are extensively used in the screening of vaccines and drugs for Cryptosporidium, but this study model does not reflect the real status of infection in immunocompetent animals. This study aimed to provide an optimized animal model for future studies of Cryptosporidium vaccine. METHODS AND RESULTS: Three mouse strains (ICR, BALB/c and KM) with or without immunosuppression were compared after challenge with Cryptosporidium tyzzeri (C tyzzeri). The results indicated that ICR mice shed a greater number of faecal oocysts (20 346 ± 203 oocysts/g) compared with BALB/c (2077 ± 142 oocysts/g) and KM mice (3207 ± 431 oocysts/g) after experimental infection with C tyzzeri (P < .001). However, ICR mouse model is uniquely effective for C tyzzeri, not for other Cryptosporidium spp. such as C parvum. ICR mice were then used to determine the immunoreactions and immunoprotection of P23-DNA vaccine (pVAX1-P23) to C tyzzeri experimental infection. The results showed that a significant increase in anti-P23 antibody levels was induced by the pVAX1-P23 vaccine. Compared to pVAX1, TB and blank control mice, pVAX1-P23 immunized mice produced specific spleen cell proliferation as well as enhanced IL-5, IL-12p70 and IFN-γ production in sera. After challenge with 5 × 106 C tyzzeri oocysts, the oocyst shedding of the pVAX1-P23 immunized group was reduced by 69.94% comparing to the infection control. CONCLUSION: These results provide an optimized animal model for the study of prophylactic vaccines and this model might be applied to other candidates against Cryptosporidium, not only for pVAX1-P23.

Seroprevalence of antibodies against Chlamydia trachomatis and enteropathogens and distance to the nearest water source among young children in the Amhara Region of Ethiopia.

The transmission of trachoma, caused by repeat infections with Chlamydia trachomatis, and many enteropathogens are linked to water quantity. We hypothesized that children living further from a water source would have higher exposure to C. trachomatis and enteric pathogens as determined by antibody responses. We used a multiplex bead assay to measure IgG antibody responses to C. trachomatis, Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae in eluted dried blood spots collected from 2267 children ages 0-9 years in 40 communities in rural Ethiopia in 2016. Linear distance from the child's house to the nearest water source was calculated. We derived seroprevalence cutoffs using external negative control populations, if available, or by fitting finite mixture models. We used targeted maximum likelihood estimation to estimate differences in seroprevalence according to distance to the nearest water source. Seroprevalence among 1-9-year-olds was 43% for C. trachomatis, 28% for S. enterica, 70% for E. histolytica, 54% for G. intestinalis, 96% for C. jejuni, 76% for ETEC and 94% for C. parvum. Seroprevalence increased with age for all pathogens. Median distance to the nearest water source was 473 meters (IQR 268, 719). Children living furthest from a water source had a 12% (95% CI: 2.6, 21.6) higher seroprevalence of S. enterica and a 12.7% (95% CI: 2.9, 22.6) higher seroprevalence of G. intestinalis compared to children living nearest. Seroprevalence for C. trachomatis and enteropathogens was high, with marked increases for most enteropathogens in the first two years of life. Children living further from a water source had higher seroprevalence of S. enterica and G. intestinalis indicating that improving access to water in the Ethiopia's Amhara region may reduce exposure to these enteropathogens in young children.

Investigation of Cryptosporidium spp. Antigen By ELISA in Stool Specimens Sent to Our Laboratory between 2010 and 2018.

OBJECTIVE: Cryptosporidium is an enteric protozoan parasite that affects human and many animal species in worldwide. Staining methods or stool antigen detecting methods are using for detection of Cryptosporidium in faeces. It is known that the ELISA method has high sensitivity and specificity in practice. The aim of this study is to demonstrate the frequency of this parasite by ELISA in samples sent to our laboratory between 2010 January and 2018 September. METHODS: The study was conducted on a total of 723 patients, 431 men and 292 women, who were referred to the Parasitology Laboratory from various outpatient clinics due to digestive system complaints. The presence of Cryptosporidium spp. antigen was investigated by ELISA method. RESULTS: Cryptosporidium spp. was not found in any patient with Nativ-lugol method, whereas Cryptosporidium spp. antigen positivity was detected in 2.8% of 723 patients. In the study, 2.5% of the males and 3.1% of the females were found positive in terms of having the parasite, and there was no significant difference in gender between the parasite frequencies. The highest rate of parasite positivity (4.5%) was found in the 0-6 age group. CONCLUSION: The high rate of cryptosporidiosis detected in our study is thought to be related to factors such as widespread animal husbandry in our region, poor hygiene rules and low socio-economic level. As a result of considering the findings of our study, evaluation of patients with intestinal complaints in terms of Cryptosporidium will be useful for accurate diagnosis, regardless of whether they have diarrhea and/or they are immunocompromised.

Detecting Cryptosporidium in Stool Samples Submitted to a Reference Laboratory.

When considering methods of detecting Cryptosporidium in patient samples, clinical and public health laboratories have historically relied primarily on microscopy. However, microscopy is time intensive and requires trained personnel to accurately identify pathogens that are present. Even with skilled analysts, the parasitemia level has the potential to fall below the level of detection. In addition, public health laboratories do not always receive specimens in fixatives that are compatible with the desired microscopic method. Antigen-based and molecular methods have proven to be effective at identifying Cryptosporidium at low levels and require less training and hands-on time. Here, we have developed and validated a real-time polymerase chain reaction (RT-PCR) laboratory-developed test (LDT) that identifies Cryptosporidium hominis and Cryptosporidium parvum, and also includes detection at the genus level to identify additional species that occasionally cause disease in humans. Results of the molecular test were compared with those obtained from modified acid-fast microscopy, immunofluorescent microscopy, an antigen-based detection rapid test, and a commercial gastrointestinal panel (GI panel). Of 40 positive samples, microscopy and antigen-based methods were able to detect Cryptosporidium in only 20 and 21 samples, respectively. The GI panel detected 33 of the 40 positive samples, even though not all specimens were received in the recommended preservative. The LDT detected Cryptosporidium in all 40 positive samples. When comparing each method for the detection of Cryptosporidium, our results indicate the LDT is an accurate, reliable, and cost-effective method for a clinical public health reference laboratory.

Cryptosporidium Diagnostic Assays: Microscopy.

Stained microscopy of fecal smears was the cornerstone of Cryptosporidium diagnosis for many years, and still provides a low-cost method for detecting oocysts. The development and commercialization of improved enzyme immunosorbent assays (EIA) for coproantigen detection provided an automatable method for mass testing, and rapid diagnostics when incorporated onto a cartridge format. Similarly, immunochromatographic lateral flow assays (ICLF) enable rapid diagnostics. Nevertheless, it is important that positive reactions by EIA or ICLF are confirmed. Here we describe microscopical methods using tinctorial stains for the diagnosis of acute cryptosporidiosis, and using immunofluorescent reagents for diagnosis or for confirmation of EIA or ICLF positive reactions.

Measuring Cryptosporidium Serologic Responses by Multiplex Bead Assay.

For more than 35 years, various assay formats have been used to detect Cryptosporidium-specific antibodies in human and animal sera. Cryptosporidium parvum 17- and 27-kDa antigens, identified from invasive sporozoites, have been used in serologic antibody assays to identify individuals infected in outbreaks of diarrheal disease caused by this protozoan parasite and to monitor exposures in communities. During infection, immunoglobulin (Ig) A, IgM, and IgG responses are elicited by these immunodominant antigens, and the parasite-specific Ig responses diminish following the resolution of infection. Using the recombinant forms of the 17- and 27-kDa C. parvum antigens and the relatively recently developed multiplex bead assay (MBA), data from serologic antibody responses can be economically and efficiently acquired, especially when the Cryptosporidium assays are integrated with assays for antibody responses to antigens from other pathogens monitored in community-wide or nation-wide serosurveys. Here we describe the coupling of the C. parvum recombinant antigens to carboxylated polystyrene beads, the data acquisition and analysis of IgG antibodies bound to the coupled beads, and the quality control methods required for data validation using the Luminex/MBA system.

Production and Purification of Functional Cryptosporidium Glycoproteins by Heterologous Expression in Toxoplasma gondii.

Development of an effective vaccine against cryptosporidiosis is a medical and veterinary priority. However, many putative Cryptosporidium vaccine candidates such as surface and apical complex antigens are posttranslationally modified with O- and N-linked glycans. This presents a significant challenge to understanding the functions of these antigens and the immune responses to them. Isolation of large amounts of native antigen from Cryptosporidium oocysts is expensive and is only feasible for C. parvum antigens. Here, we describe a method of producing recombinant, functional Cryptosporidium glycoprotein antigens in Toxoplasma gondii. These functional recombinant proteins can be used to investigate the role of glycotopes in Cryptosporidium immune responses and parasite-host cell interactions.

An immunocompetent rat model of infection with Cryptosporidium hominis and Cryptosporidium parvum.

A major obstacle to developing vaccines against cryptosporidiosis, a serious diarrheal disease of children in developing countries, is the lack of rodent models essential to identify and screen protective immunogens. Rodent models commonly used for drug discovery are unsuitable for vaccine development because they either are purposefully immunodeficient or immunosuppressed. Here, we describe the development and optimization of an immunocompetent intratracheal (IT) rat model susceptible to infections with sporozoites of Cryptosporidium parvum and Cryptosporidium hominis - the primary causes of human cryptosporidiosis. A model suitable for screening of parasite immunogens is a prerequisite for immunogen screening and vaccine development.

Are immunoenzymatic tests for intestinal protozoans reliable when used on archaeological material?

Intestinal protozoans found in ancient human samples have been studied primarily by microscopy and immunodiagnostic assays. However, such methods are not suitable for the detection of zoonotic genotypes. The objectives of the present study were to utilize immunoenzimatic assays for coproantigen detection of Cryptosporidium sp., Giardia duodenalis, and Entamoeba histolytica/Entamoeba dispar in sixty ancient human and animal samples collected from 14 archaeological sites in South America, and to carry out a critical analysis of G. duodenalis according to results obtained from three diagnostic methodologies: microscopy, immunodiagnostic tests (immunoenzymatic and immunofluorescence), and molecular biology (PCR and sequencing). More than half (31/60) of the samples analyzed using immunoenzymatic tests were positive for at least one of the intestinal protozoans, with 46.6% (28/60) corresponding to G. duodenalis, 26.6% (16/60) to Cryptosporidium sp., and 5% (3/60) to E. histolytica/E. dispar. Cryptosporidium sp. and G. duodenalis coinfection was observed in 15% (9/60) of the samples, whereas all three protozoans were found in 5% (3/60) of samples. In the Northeast Region of Brazil, by immunoenzymatic tests there is evidence that G. duodenlais and Cryptosporidium sp. have infected humans and rodents for at least 7150 years. However, for G. duodenalis, the results from the three diagnostic tests were discordant. Specifically, despite the efficiency of the molecular biology assay in the experimental models, G. duodenalis DNA could not be amplified from the ancient samples. These results raise the following question: Are all ancient samples positive for coproantigen of G. duodenalis by immunoenzymatic tests truly positive? This scenario highlights the importance of further studies to evaluate the sensitivity and specificity of the immunoenzymatic method in the archaeological context.

A Genetically Tractable, Natural Mouse Model of Cryptosporidiosis Offers Insights into Host Protective Immunity.

Cryptosporidium is a leading cause of diarrheal disease and an important contributor to early childhood mortality, malnutrition, and growth faltering. Older children in high endemicity regions appear resistant to infection, while previously unexposed adults remain susceptible. Experimental studies in humans and animals support the development of disease resistance, but we do not understand the mechanisms that underlie protective immunity to Cryptosporidium. Here, we derive an in vivo model of Cryptosporidium infection in immunocompetent C57BL/6 mice by isolating parasites from naturally infected wild mice. Similar to human cryptosporidiosis, this infection causes intestinal pathology, and interferon-γ controls early infection while T cells are critical for clearance. Importantly, mice that controlled a live infection were resistant to secondary challenge and vaccination with attenuated parasites provided protection equal to live infection. Both parasite and host are genetically tractable and this in vivo model will facilitate mechanistic investigation and rational vaccine design.

Tandem Orthotopic Living Donor Liver Transplantation Followed by Same Donor Haploidentical Hematopoietic Stem Cell Transplantation for DOCK8 Deficiency.

BACKGROUND: An 11-year-old girl with dedicator of cytokinesis 8 (DOCK8) deficiency was proposed for potentially curative hematopoietic stem cell transplantation (HSCT), the donor being her haploidentical mother. However, end-stage liver disease caused by chronic Cryptosporidium infection required liver transplantation before HSCT. METHODS: Consequently, a staged approach of a sequential liver transplant followed by a HSCT was planned with her mother as the donor for both liver and HSCT. RESULTS: The patient successfully underwent a left-lobe orthotopic liver transplant; however, she developed a biliary leak delaying the HSCT. Notably, the recipient demonstrated 3% donor lymphocyte chimerism in her peripheral blood immediately before HSCT. Haploidentical-related donor HSCT performed 2 months after liver transplantation was complicated by the development of acyclovir-resistant herpes simplex virus viremia, primary graft failure, and sinusoidal obstruction syndrome. The patient died from sinusoidal obstruction syndrome-associated multiorgan failure with Candida sepsis on day +40 following HSCT. CONCLUSIONS: We discuss the many considerations inherent to planning for HSCT preceded by liver transplant in patients with primary immunodeficiencies, including the role of prolonged immunosuppression and the risk of infection before immune reconstitution. We also discuss the implications of potential recipient sensitization against donor stem cells precipitated by exposure of the recipient to the donor lymphocytes from the transplanted organ.

Rapid diagnostic tests relying on antigen detection from stool as an efficient point of care testing strategy for giardiasis and cryptosporidiosis? Evaluation of a new immunochromatographic duplex assay.

Microscopy is the gold standard for the diagnosis of gastrointestinal parasites but is time-consuming and dependent on operator skills. Rapid diagnostic tests represent alternative methods but most evaluations have been conducted on a limited number of samples preventing their implementation in the clinical setting. We evaluated a new CE-IVD marked immunochromatographic assay (Crypto/Giardia K-SeT®, Coris Bioconcept) for the detection of G. intestinalis and Cryptosporidium spp. in 2 phases (retrospective and prospective) on a set of 482 stool samples including rare Cryptosporidium species. Besides G. intestinalis, this test could represent a rapid and reliable alternative to the modified Ziehl-Neelsen staining for the diagnosis of cryptosporidiosis (sensitivity/specificity were 89.2%/99.3% and 86.7%/100% for G. intestinalis and Cryptosporidium resp.), reducing diagnostic delays. Such strategy would also be time-saving by avoiding wet mount microscopy and concentrations steps, being particularly appropriate for laboratories having little expertise in microscopy or not able to implement molecular diagnostic methods.

Identification of novel vaccine candidates against cryptosporidiosis of neonatal bovines by reverse vaccinology.

The apicomplexan protozoan Cryptosporidium parvum is an important causative agent of diarrhea of neonatal bovines. Vaccination has been proposed as an advantageous strategy against cryptosporidiosis of calves since besides protection against disease it has also the potential to prevent dissemination of infective oocysts into the environment. Antigens anchored to the parasite surface via glycosylphosphatidylinositol (GPI) are implicated in host cell attachment and invasion and represent promising vaccine candidates. A reverse vaccinology approach was employed to (i) identify the GPI-anchored proteome of C. parvum using available web-based bioinformatic tools and (ii) characterize previously unrecognized novel vaccine antigens. Altogether, 14 putative GPI-anchored proteins could be determined of which CpH1 and CpSUB2 as well as GP60 were further characterized. Sequencing and comparison of GP60, CpH1, and CpSUB1 alleles amplified from different geographic isolates showed a high degree of conservation. All three antigens were recombinant expressed and immunoblotted using sera of 12 Cryptosporidium-infected calves sampled at age periods 1-11 and 12-28 days after birth. Specific antibody reactions against the studied antigens were detected in all analyzed calves, demonstrating their immunreactivity and expression, and recognition in vivo at an early stage of host infection. Besides the acknowledged GP60 vaccinogen, the presented reverse vaccinology approach reveals the additional vaccine candidates CpH1 and CpSUB1 for inclusion into a subunit vaccine formulation.