In immunocompromised patients with primary or acquired immune deficiency (solid organ or bone marrow transplant recipients), cryptosporidiosis may cause severe diseases, sometimes complicated with biliary duct or lung location, resulting in long-term morbidity and a non-negligible mortality reaching 6 to 8%. Nevertheless, clinicians are faced with a limited therapeutic arsenal based on recommendations with a low level of evidence due the lack of clinical trials. Diverse therapeutic modalities have been studied in case reports, case series and clinical trials in different populations mainly in HIV patients with low CD4-Tcells<200/mm3. The aim of the presentation was to review a total of 21 cases of immunocompromised patients with severe cryptosporidiosis. We report the experience of the weekly Multidisciplinary Concertation Meetings (RCP) from November 2023 to november 2024 set up by the Reference National Center for Cryptosporidiosis, Microsporidia and Other Digestive Protozoa (CMAP). In 12 months, RCP was approached for 21 immunocompromised patients. Fourteen patients were solid organ transplant recipients (10 kidney, 2 lung, 2 liver), 2 had primary immunodeficiency (1 LOCID, 1 CD40L deficiency), 4 were bone marrow transplant patients (2 B lymphid leukemia, 1 chronic myeloid leukemia, 1 SCID with IFR4 deficiency) and 1 had mastocytosis treated with tacrolimus. The mean age of the adults was 42.5±14.3 years. Among these 21 patients, 4 children had cryptosporidiosis (7 and 4 years, kidney transplant recipients; 12 and 3.5 years, hematopoietic bone marrow transplant). All patients presented with deterioration of their general condition with weight loss and renal failure. Four patients developed biliary complications or even sclerosing cholangitis. One bone marrow transplanted child died with disseminated cryptosporidiosis. Species identification, when available resulted in mainly C. parvum (9 cases ; IIdA19G1, IIaA21G1R1, IIaA15G2R1, IIaA19G2R1, IIdA22G1, IIcA5G3) but also C. hominis (2 ; IbA10G2, IdA16), C. felis (1) and C. ubiquitum (1). The RCP's opinion allowed a therapeutic adaptation concerning the dosages and the use of molecules sometimes not known by the requesting clinicians. Globally, we were sollicitated ~ 1 months after the beginning of symptoms, and after the failure of a first line treatment. The RCPs also made it possible to specify the strategy for monitoring/parasitological control of patients during and after treatment. Combination therapy with multiple drugs appearing to be more effective than monotherapy with a single antiparasitic agent, and most patients have been treated with combinations of nitazoxanide with azithromycin for Cryptosporidium spp infections or other agents (for treatment durations ranging from 2 weeks to several months). Because cell-mediated immunity plays a key role in host defense, efforts were made to minimize immunosuppression in transplant recipients. The RCP's opinion allowed a therapeutic adaptation concerning the dosages and the use of molecules sometimes not known by the requesting clinicians. Globally, we were sollicitated ~ 1 months after the beginning of symptoms, and after the failure of a first line treatment. The RCPs also made it possible to specify the strategy for monitoring/parasitological control of patients during and after treatment. Combination therapy with multiple drugs appearing to be more effective than monotherapy with a single antiparasitic agent, and most patients have been treated with combinations of nitazoxanide with azithromycin for Cryptosporidium spp infections or other agents (for treatment durations ranging from 2 weeks to several months). Because cell-mediated immunity plays a key role in host defense, efforts were made to minimize immunosuppression in transplant recipients.

Giardia duodenalis causes giardiasis, a major worldwide diarrheal disease in humans.  Treatment of giardiasis relies mainly on metronidazole and albendazole (ABZ) and the emergence of parasites resistant to these agents has stimulated studies on the molecular mechanisms underlying this phenomenon. In our group we have obtained trophozoite cultures of the WB strain of this parasite displaying resistance to increasing concentrations of ABZ (RA 1.35, 8 and 250 µM) that were used as models of ABZ resistance in Giardia. In recent studies, we observed that the addition of the iron-containing cofactor heme to Giardia trophozoite cultures lowered the IC50 of ABZ-resistant clones, suggesting that heme-containing enzymes, particularly the nitric oxide (NO)-detoxifying enzyme flavohemoglobin (gFlHb) could be involved in ABZ resistance in this parasite. Molecular docking and dynamics analyses predicted that gFlHb interacts with ABZ and its metabolites ABZSO and ABZSOO. Recombinant gFlHb was expressed, its spectrometry profile was analyzed and its NADH oxidase activity was determined in the presence of ABZ. LC-MS/MS analyses confirmed that gFlHb metabolizes ABZ producing ABZSO but produces an excess of superoxide anion that might be toxic in a cellular context. Also, the expression levels of gFlHb in ABZ-susceptible WB strain and ABZ-resistant (RA8 and RA250) trophozoites were decreased in ABZ-resistant clones as compared to ABZ-susceptible ones. Remarkably, when ABZ-resistant trophozoites were transfected with a vector encoding gFlHb,  these trophozoites became much more susceptible to ABZ, suggesting that gFlHb is involved in both ABZ metabolism and ABZ-resistance through a passive pharmacokinetic mechanism. In further studies we analyzed the participation of epigenetic regulators such as sirtuins in ABZ-resistant cultures. Initially we analyzed the expression of sirtuins in the three ABZ-resistant Giardia. Our results demonstrated a significant upregulation of sirtuins gdSir2.1, gdSir2.2 and gdSir2.3 in all ABZ-resistant lines. Using the CRISPRi strategy to repress several sirtuins, we observed that knockdown of gdSir2.1 and gdSir2.3 in Giardia trophozoites resulted in heightened susceptibility to both ABZ and hydrogen peroxide. Additional studies suggest that sirtuins contribute to the regulation of reactive oxygen species (ROS) levels, oxidative DNA damage, and the expression of oxidative stress response genes. In knockdown strains downregulation of gdSir2.1 decreases the expression of Trx, Prx and SOR, while downregulation of gdSir2.3 decreases the expression of Fdp, Prx and NADHox in the parasite. Collectively, our results demonstrated that gdSir2.1 and gdSir2.3 play a significant role in regulation of ABZ-resistance, primarily through the modulation of parasite´s oxidative stress response.

Giardia lamblia is a globally prevalent protozoan parasite, responsible for substantial diarrheal morbidity and mortality worldwide. While typically presenting as an acute illness, giardiasis can evolve into a chronic condition, highlighting the importance of understanding the immune mechanisms involved in both the pathogenesis and recovery process to guide vaccine and therapeutic development. In this study, 21 healthy controls and 21 patients with confirmed Giardia infection, primarily due to travel-related exposure, were included. Patients were not hospitalized, but suffered from clinical symptoms including abdominal cramps and diarrhea. Plasma samples were collected at the time of diagnosis and 6 weeks after successful treatment. Plasma samples were analyzed for 96 protein markers of inflammation using Proximity Extension Assay (Olink Target 96 Inflammation) and ELISA. Marker levels were compared across the two timepoints using the Wilcoxon signed-rank test and against healthy controls using the Mann-Whitney U test with multiple-test corrections. Our results show significantly elevated plasma levels of inflammatory markers in Giardia infection, including CCL11, CCL20, CCL25, CCL28, CXCL9, FGF-21, IL-10, IL-12B, IL-17A/C, IL-18, IL-8, MMP-10, TNF and TNFRSF9, compared to levels in the same patients six weeks after successful treatment was initiated. Giardia infected patients also exhibited elevated levels to these and some more inflammation markers such as IFNgamma, PD-L1, TGFbeta, MMP-1 and Oncostatin M (OSM) compared to healthy controls. No significant differences were observed between recovered patients and healthy controls. The marker profile suggest a mixed Th1/Th17-mediated immune response, characterized by increased IFNgamma, IL-18, IL-12B and IL-17A/C. CCL20 is involved in the formation and function of mucosal lymphoid tissues and attracts lymphocytes and dendritic cells. Elevated CCL28 and CXCL9 suggest increased recruitment of B and T cells to mucosal tissues, and also of eosinophils as CCL11 (eotaxin-1) was elevated compared to healthy controls. Notable increases in markers of epithelial repair (MMP-10, IL-17A/C) and markers of regulatory immune responses (IL-10, PD-L1, TNFRSF9) were observed, likely mitigating inflammation. Furthermore, growth factors and tissue repair markers such as VEGFA, HGF and OSM were upregulated, alongside several matrix metalloproteinases involved in extracellular matrix remodeling.

In conclusion, acute Giardia infection elicits a robust pro-inflammatory immune response, characterized by Th1/Th17-mediated immunity, tissue repair, and regulatory mechanisms. These findings offer critical insights into host-pathogen interactions and may inform future strategies for therapeutics and vaccine development.

Drug resistance hampers the treatment of giardiasis, one of the world’s most common gastro-intestinal parasitic diseases of humans and animals. On an annual basis ~200 million people develop giardiasis, a disease that impacts child development and the long-term health of many adults. However, there is no vaccine for giardiasis and treatment options are failing due to multiple factors including variable activity and drug resistant parasites. Moreover, current treatment strategies are monotherapies that do little to combat the development of drug resistance. To improve this position combination therapies that include compounds with complementary pharmacodynamic and pharmacokinetic activities are needed. However, little has been done to identify best practice combination therapies for giardiasis. As a mechanism to pave the way towards the development of highly effective drug combination therapies for giardiasis we have investigated the in vitro pharmacodynamic interactions of a selection of currently used antigiardial drugs and several new investigational compounds of interest in the field. These studies have identified multiple synergistic compound combinations that may be useful in the in vivo setting including combinations of currently used drugs with new antigiardial compounds that have Interaction (I) values >3.0; P<0.05. While in vivo studies are now needed to determine if these promising in vitro interactions are transferrable to the clinical setting, taken together with pharmacokinetic data, these results are exciting and suggest that novel antigiardial combination therapies can be developed to help combat treatment refractory cases of giardiasis. Current data and planned in vivo activity investigations with the most promising compound combinations will be discussed.

Giardiasis, an intestinal infection caused by the protozoan parasite Giardia intestinalis, often poses significant treatment challenges due to emerging unresponsiveness to standard metronidazole treatment. Despite extensive research, reliable genetic markers for metronidazole resistance have remained difficult to identify. Aiming to contribute to the understanding of this complex problem, we analyzed 28 clinical isolates of G. intestinalis from sub-assemblage AII, collected from patients exhibiting varying clinical responses to metronidazole treatment. Our study focused on the copy number variation (CNV) of the multicopy flavohemoprotein gene, a potential player in drug resistance mechanisms. Utilizing digital PCR and nextgeneration sequencing (NGS), we aimed to assess the gene's CNV and explore its correlation with metronidazole resistance. Digital PCR, in particular, provided a highly sensitive and precise quantification method, allowing us to detect subtle variations in gene copy numbers that traditional techniques might overlook. We also evaluated chromosomal ploidy in 18 of these samples to understand its contribution to genomic variability. To broaden our perspective, we further examined flavohemoprotein CNV in additional 17 samples from other A and B subassemblages. Our findings revealed variable CNVs of the flavohemoprotein gene among the isolates, yet no direct correlation with clinical metronidazole resistance emerged. Significant differences in flavohemoprotein CNVs were observed across different G. intestinalis sub-assemblages, highlighting the parasite's genetic diversity. Moreover, our findings demonstrated a propensity for aneuploidy in G. intestinalis, contributing to genomic variability both within and between sub-assemblages. Importantly, we also identified a technical bias, discrepancies in CNVs detected from NGS data linked to whole-genome amplification, a limitation that digital PCR effectively overcame. These results suggest that the complexity of metronidazole resistance in G. intestinalis is influenced by multiple factors beyond flavohemoprotein CNV and aneuploidy. The absence of significant differences in flavohemoprotein CNV between resistant and sensitive isolates underscores the need for continued research to identify reliable genetic markers of resistance. Our study demonstrates that digital PCR can effectively validate and complement NGS results in analyzing CNVs. By providing more accurate CNV measurements, our work contributes to a better understanding of the genetic mechanisms underlying drug resistance in this parasite.

Giardiasis is a worldwide-spread parasitic disease caused by a single-celled eukaryotic pathogen, Giardia intestinalis. The increasing number of metronidazole refractory cases (MTZ‑TF) raises questions about the shared genetic background of parasites that would be associated with treatment failure. Despite the long time and widespread use of MTZ, the understanding of the nature of MTZ-TF is incomplete. Our study investigated Giardia genotypes, their relationship to MTZ treatment outcome, associated clinical presentations, and geographical distribution patterns in a patient cohort from the Czech Republic. A multilocus genotyping method was applied to 86 Giardia DNA isolates collected over four years to assort them to assemblages/sub-assemblages. The prevalence of MTZ-TF in this cohort was 28% (24/86), with 23 out of 24 cases imported to the Czech Republic, mostly from the Indian subcontinent (62.5%, 15/24). The genotyping revealed common human infecting assemblages only (A and B), with a predominance of assemblage B (83%, 71/86).We found an association between MTZ-TF and sub-assemblage BIII (p = 0.012), and an Indian subcontinent origin of infection (p = 0.0024). None of the followed symptoms (diarrhoea, abdominal pain, bloating, nausea, or vomiting) showed a higher prevalence or significant link to MTZ-TF. In the β-giardin gene locus, shared polymorphisms were identified among some MTZ-TF isolates (5/16) from India. However, these polymorphisms reflect their phylogenetic relationship rather than serving as a shared resistance marker. The results highlight the importance of assemblage B in MTZ-TF and suggest a higher risk of acquiring MTZ-TF giardiasis in the Indian subcontinent.