Developing and Optimizing Shrub Parameters Representing Sagebrush (\u3ci\u3eArtemisia\u3c/i\u3e spp.) Ecosystems in the Northern Great Basin Using the Ecosystem Demography (EDv2.2) Model

Ecosystem dynamic models are useful for understanding ecosystem characteristics over time and space because of their efficiency over direct field measurements and applicability to broad spatial extents. Their application, however, is challenging due to internal model uncertainties and complexities arising from distinct qualities of the ecosystems being analyzed. The sagebrush-steppe ecosystem in western North America, for example, has substantial spatial and temporal heterogeneity as well as variability due to anthropogenic disturbance, invasive species, climate change, and altered fire regimes, which collectively make modeling dynamic ecosystem processes difficult. Ecosystem Demography (EDv2.2) is a robust ecosystem dynamic model, initially developed for tropical forests, that simulates energy, water, and carbon fluxes at fine scales. Although EDv2.2 has since been tested on different ecosystems via development of different plant functional types (PFT), it still lacks a shrub PFT. In this study, we developed and parameterized a shrub PFT representative of sagebrush (Artemisia spp.) ecosystems in order to initialize and test it within EDv2.2, and to promote future broad-scale analysis of restoration activities, climate change, and fire regimes in the sagebrushsteppe ecosystem. Specifically, we parameterized the sagebrush PFT within EDv2.2 to estimate gross primary production (GPP) using data from two sagebrush study sites in the northern Great Basin. To accomplish this, we employed a three-tier approach. (1) To initially parameterize the sagebrush PFT, we fitted allometric relationships for sagebrush using field-collected data, information from existing sagebrush literature, and parameters from other land models. (2) To determine influential parameters in GPP prediction, we used a sensitivity analysis to identify the five most sensitive parameters. (3) To improve model performance and validate results, we optimized these five parameters using an exhaustive search method to estimate GPP, and compared results with observations from two eddy covariance (EC) sites in the study area. Our modeled results were encouraging, with reasonable fidelity to observed values, although some negative biases (i.e., seasonal underestimates of GPP) were apparent. Our finding on preliminary parameterization of the sagebrush shrub PFT is an important step towards subsequent studies on shrubland ecosystems using EDv2.2 or any other process-based ecosystem model

Regional Scale Dryland Vegetation Classification with an Integrated Lidar-Hyperspectral Approach

The sparse canopy cover and large contribution of bright background soil, along with the heterogeneous vegetation types in close proximity, are common challenges for mapping dryland vegetation with remote sensing. Consequently, the results of a single classification algorithm or one type of sensor to characterize dryland vegetation typically show low accuracy and lack robustness. In our study, we improved classification accuracy in a semi-arid ecosystem based on the use of vegetation optical (hyperspectral) and structural (lidar) information combined with the environmental characteristics of the landscape. To accomplish this goal, we used both spectral angle mapper (SAM) and multiple endmember spectral mixture analysis (MESMA) for optical vegetation classification. Lidar-derived maximum vegetation height and delineated riparian zones were then used to modify the optical classification. Incorporating the lidar information into the classification scheme increased the overall accuracy from 60% to 89%. Canopy structure can have a strong influence on spectral variability and the lidar provided complementary information for SAM’s sensitivity to shape but not magnitude of the spectra. Similar approaches to map large regions of drylands with low uncertainty may be readily implemented with unmixing algorithms applied to upcoming space-based imaging spectroscopy and lidar. This study advances our understanding of the nuances associated with mapping xeric and mesic regions, and highlights the importance of incorporating complementary algorithms and sensors to accurately characterize the heterogeneity of dryland ecosystems

Evaluation of a Novel Commercial Real-Time PCR Assay for the Simultaneous Detection of Cryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica

Thorough independent assessment of the diagnostic performance of novel diagnostic assays is essential to ascertain their true usefulness and applicability in routine clinical practice. This is particularly true for commercially available kits based on multiplex real-time PCR aimed to detect and differentiate multiple pathogens in a single biological sample. Cryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica are the most common diarrhea-causing protozoan species globally. Misdiagnosis is a concern for asymptomatic and chronic infections. Multiplexing, i.e., the detection of more than one parasite in a single test by real-time PCR, allows high diagnostic performance with favorable cost-effectiveness. We conducted a clinical evaluation of the VIASURE Cryptosporidium, Giardia, & E. histolytica real-time PCR assay (CerTest Biotec, San Mateo de Gallego, Spain) against a large panel (n = 358) of well-characterized DNA samples positive for Cryptosporidium spp. (n = 96), G. duodenalis (n = 115), E. histolytica (n = 25), and other parasitic species of the phyla Amoebozoa (n = 11), Apicomplexa (n = 14), Euglenozoa (n = 8), Heterokonta (n = 42), Metamonada (n = 37), Microsporidia (n = 4), and Nematoda (n = 6). DNA samples were obtained from clinical stool specimens or cultured isolates in a national reference center. Estimated sensitivity and specificity were 0.96 and 0.99 for Cryptosporidium spp., 0.94 and 1 for G. duodenalis, and 0.96 and 1 for E. histolytica, respectively. Positive and negative predictive values were calculated as 1 and 0.98 for Cryptosporidium spp., 0.99 and 0.98 for G. duodenalis, and 1 and 0.99 for E. histolytica, respectively. The assay identified six Cryptosporidium species (Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium scrofarum, and Cryptosporidium ryanae) and four G. duodenalis assemblages (A, B, C, and F). The VIASURE assay provides rapid and accurate simultaneous detection and identification of the most commonly occurring species and genetic variants of diarrhea-causing parasitic protozoa in humans. IMPORTANCE Thorough independent assessment of the diagnostic performance of novel diagnostic assays is essential to ascertain their true usefulness and applicability in routine clinical practice. This is particularly true for commercially available kits based on multiplex real-time PCR aimed to detect and differentiate multiple pathogens in a single biological sample. In this study, we conducted a clinical evaluation of the VIASURE Cryptosporidium, Giardia, & E. histolytica real-time PCR assay (CerTest Biotec) for the detection and identification of the diarrhea-causing enteric protozoan parasites Cryptosporidium spp., G. duodenalis, and E. histolytica. A large panel of well-characterized DNA samples from clinical stool specimens or cultured isolates from a reference center was used for this purpose. The VIASURE assay demonstrated good performance for the routine testing of these pathogens in clinical microbiological laboratories

Molecular Detection and Genotyping of Enteric Protists in Asymptomatic Schoolchildren and Their Legal Guardians in Madrid, Spain

Asymptomatic carriage of diarrhoea-causing enteric protist parasites in the general population is poorly understood, particularly in medium- to high-income countries. This molecular epidemiological survey investigates the presence, molecular diversity, and household transmission of Giardia duodenalis, Cryptosporidium spp., Blastocystis sp., and Enterocystozoon bieneusi in schoolchildren aged 2–13 years (n = 74) and their legal guardians (n = 6) in Madrid, Spain. Enteroparasite detection and genotyping was conducted in stool samples by molecular (PCR and Sanger sequencing) methods. Potential associations linked to infections were investigated through epidemiological questionnaires. Giardia duodenalis was the most prevalent enteric parasite found (14%, 95% CI: 7.1–23), followed by Blastocystis sp. (10%, 95% CI: 6.2–22) and Cryptosporidium spp. (3.8%, 95% CI: 0.78–11). None of the participants tested positive for E. bieneusi. Sequence analyses revealed the presence of G. duodenalis assemblage B, sub-assemblage BIV in a single child. The three Cryptosporidium isolates obtained were assigned to C. hominis, two of them belonging to the gp60 subtype IbA10G2. Four Blastocystis subtypes were identified including ST2 (38%, 3/8), ST3 (25%, 2/8), ST4 (25%, 2/8), and ST8 (12%, 1/8). All G. duodenalis and Cryptosporidium isolates were detected in children only. Blastocystis ST3 and ST4 were circulating in members of the same household. Blastocystis carriage rates increased with the age of the participants. Presence of diarrhoea-causing enteric protists was common in apparently healthy children.This research was funded by Health Institute Carlos III (ISCIII), Ministry of Science, Innovation and Universities, Spain, grant number PI16CIII/00024.S

Detection of enteric parasites and molecular characterization of Giardia duodenalis and Blastocystis sp. in patients admitted to hospital in Ankara, Turkey.

This epidemiological study assesses the occurrence of enteric parasites in 4303 patients attended at two public hospitals in Ankara (Turkey) during 2018-2019. Microscopy was used as a screening test. Giardia duodenalis was also identified using a commercial ELISA for the detection of parasite-specific coproantigens. Giardia-positive samples by microscopy/ELISA were confirmed by real-time PCR and characterized using a multilocus genotyping scheme. Blastocystis sp. was genotyped in a sample subset. Blastocystis sp. (11.1%, 95% CI 11.4‒14.8%) and G. duodenalis (1.56%, 95% CI 1.22‒1.96) were the most prevalent pathogens found. Cryptosporidium spp., Entamoeba histolytica and intestinal helminths were only sporadically (<0.5%) found. For G. duodenalis, sequence (n = 30) analyses revealed the presence of sub-assemblages AII (23.3%), discordant AII/AIII (23.3%) and mixed AII + AIII (6.7%) within assemblage A, and BIII (10.0%), BIV (3.3%) and discordant BIII/BIV (23.3%) within assemblage B. Two additional sequences (6.7%) were assigned to the latter assemblage but sub-assemblage information was unknown. No associations between G. duodenalis assemblages/sub-assemblages and sociodemographic and clinical variables could be demonstrated. For Blastocystis sp., sequence (n = 6) analyses identified subtypes ST1, ST2 and ST3 at equal proportions. This is the first molecular characterization of G. duodenalis based on MLG conducted in Turkey to date.This research was partially funded by the Scientific Research Unit of Gazi University (Ankara, Turkey) under project number 01/2017-15, and by the Health Institute Carlos III (ISCIII), Ministry of Science, Innovation and Universities (Spain) under grant number PI16CIII/00024N

Molecular Diversity of Giardia duodenalis, Cryptosporidium spp., and Blastocystis sp. in Symptomatic and Asymptomatic Schoolchildren in Zambézia Province (Mozambique).

Infections by the protist enteroparasites Giardia duodenalis, Cryptosporidium spp., and, to a much lesser extent, Blastocystis sp. are common causes of childhood diarrhoea in low-income countries. This molecular epidemiological study assesses the frequency and molecular diversity of these pathogens in faecal samples from asymptomatic schoolchildren (n = 807) and symptomatic children seeking medical attention (n = 286) in Zambézia province, Mozambique. Detection and molecular characterisation of pathogens was conducted by polymerase chain reaction (PCR)-based methods coupled with Sanger sequencing. Giardia duodenalis was the most prevalent enteric parasite found [41.7%, 95% confidence interval (CI): 38.8‒44.7%], followed by Blastocystis sp. (14.1%, 95% CI: 12.1‒16.3%), and Cryptosporidium spp. (1.6%, 95% CI: 0.9‒2.5%). Sequence analyses revealed the presence of assemblages A (7.0%, 3/43) and B (88.4%, 38/43) within G. duodenalis-positive children. Four Cryptosporidium species were detected, including C. hominis (30.8%; 4/13), C. parvum (30.8%, 4/13), C. felis (30.8%, 4/13), and C. viatorum (7.6%, 1/13). Four Blastocystis subtypes were also identified including ST1 (22.7%; 35/154), ST2 (22.7%; 35/154), ST3 (45.5%; 70/154), and ST4 (9.1%; 14/154). Most of the genotyped samples were from asymptomatic children. This is the first report of C. viatorum and Blastocystis ST4 in Mozambique. Molecular data indicate that anthropic and zoonotic transmission (the latter at an unknown rate) are important spread pathways of diarrhoea-causing pathogens in Mozambique.This research was funded by the Health Institute Carlos III (ISCIII), Ministry of Economy and Competitiveness (Spain), grant number PI16CIII/00024.S

Long-Term Preservation and Storage of Faecal Samples in Whatman® Cards for PCR Detection and Genotyping of Giardia duodenalis and Cryptosporidium hominis

Preservation and conservation of biological specimens, including faecal samples, is a challenge in remote areas or poor-resource settings where the cold chain cannot be maintained. This study aims at evaluating the suitability of filter cards for long-term storage of faecal samples of animal and human origin positive to the diarrhoea-causing protozoan parasites, Giardia duodenalis and Cryptosporidium hominis. Three commercially available Whatman® Filter Cards were comparatively assessed: the FTA® Classic Card, the FTA® Elute Micro Card, and the 903 Protein Saver Card. Human faecal samples positive to G. duodenalis (n = 5) and C. hominis (n = 5) were used to impregnate the selected cards at given storage (1 month, 3 months, and 6 months) periods and temperature (-20 °C, 4 °C, and room temperature) conditions. Parasite DNA was detected by PCR-based methods. Sensitivity assays and quality control procedures to assess suitability for genotyping purposes were conducted. Overall, all three Whatman® cards were proven useful for the detection and molecular characterisation of G. duodenalis and C. hominis under the evaluated conditions. Whatman® cards represent a simple, safe, and cost-effective option for the transportation, preservation, and storage of faecal samples without the need of the cold chain.This research was funded by the Health Institute Carlos III (ISCIII), Ministry of Science, Innovation and Universities (Spain), grant number PI16CIII/00024. David González-Barrio was recipient of a “Sara Borrell” postdoctoral fellow-ship (CD19CIII/00011) funded by the Spanish Ministry of Science, Innovation and Universities.S

Prevalence and public health relevance of enteric parasites in domestic dogs and cats in the region of Madrid (Spain) with an emphasis on Giardia duodenalis and Cryptosporidium sp

Background: Pet dogs and cats exert an unquestionable beneficial effect in the well-being of their owners, but can also act as a source of zoonotic infections if improperly cared. Objectives: We investigated the occurrence, risk factors, genetic variability and zoonotic potential of intestinal parasites in dogs and cats attended in a clinical veterinary setting in Spain. Methods: Canine (n = 252) and feline (n = 35) faecal samples were collected during 2017-2019 and analysed by coproparasitological methods. A rapid lateral immunochromatographic test (ICT) was used for detecting Giardia duodenalis and Cryptosporidium sp. Samples positive at microscopy examination and/or ICT were reassessed by molecular methods. Results: Overall, 48.8% (123/252) of dogs and 48.6% (17/35) of cats were infected by enteric parasites. In dogs, G. duodenalis was the most prevalent species (40.9%), followed by Cystoisospora sp. (7.1%), and Toxocara canis (5.2%). In cats, Joyeuxiella sp. and Toxocara cati were the dominant species (20.0% each), followed by G. duodenalis (14.3%), D. caninum (5.7%) and Cystoisospora felis and Toxascaris leonina (2.9% each). Pups and kittens were more likely to harbour intestinal parasites and develop clinical signs. Sequence analyses of dog isolates revealed the presence of assemblages A (n = 1), C (n = 4), D (n = 4) and C+D (n = 1) within G. duodenalis; C. parvum (n = 1) and C. canis (n = 4) within Cryptosporidium and PtEb IX (n = 1) in Enterocytozoon bieneusi. A novel C. canis subtype family, named XXi, is reported. Conclusions: Our results highlight that (i) well-cared dogs carry zoonotic enteric protozoan parasites of public health relevance, (ii) proper hygiene practices and routine veterinary treatment are essential to prevent zoonotic infections, (iii) vulnerable populations should avoid contact with pups/kittens with diarrhoea and (iv) infected dogs might be major contributors to the environmental contamination with soil-transmitted helminths (STHs) eggs.This study was partially funded by the Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness under project PI19CIII/00029, and by Alfonso X el Sabio Foundation under proyect 1.011.019 (Grant / Award Number).S

Co-Infection with Cryptosporidium meleagridis and Enterocytozoon bieneusi in an HIV+ Colombian Patient

A 44-year-old human immunodeficiency virus-infected (HIV+) female with severe immunodeficiency Category 3 (C3) diagnosed in 2010 was admitted to hospital with acute diarrhoea. She was non-adherent to antiretroviral therapy (ART) and had a previous suspicion of respiratory symptoms with a cough that had been persisting for 15 days. Clinical examination revealed severe immune deterioration (viral load: 109,655 copies/mL; CD4+ count: 14 cells/mm3), respiratory symptoms (negative sputum Gram stain and tuberculosis culture), and neurological deterioration (serological assays negative for Cryptococcus spp. and Toxoplasma gondii). A coproculture was negative for Campylobacter spp., Salmonella spp., and Shigella spp. Ziehl–Neelsen staining of faecal smears revealed the presence of Cryptosporidium spp. oocysts. PCR testing and sequencing confirmed a concomitant infection with C. meleagridis and Enterocytozoon bieneusi. The patient was treated with metronidazole (500 mg every 8 h for 5 days) and nitazoxanide (500 mg every 12 h for 14 days). After requesting voluntary discharge and abandoning ART and parasiticidal treatments, she experienced a dramatic deterioration of her state of health and contact with her was lost. Our results have demonstrated that molecular-based testing improves the detection of opportunistic pathogens that are difficult to detect by routine microscopy, allows for transmission dynamics investigations, and assists in choosing the best chemotherapeutical option.This research was funded by the Health Institute Carlos III (ISCIII) and the Ministry of Economy and Competitiveness (Spain) under project PI16CIII/00024.S

High Diversity of Giardia duodenalis Assemblages and Sub-Assemblages in Asymptomatic School Children in Ibadan, Nigeria

Giardia duodenalis is a significant contributor to the burden of diarrheal disease in sub-Saharan Africa. This study assesses the occurrence and molecular diversity of G. duodenalis and other intestinal parasites in apparently healthy children (n = 311) in Ibadan, Nigeria. Microscopy was used as a screening method and PCR and Sanger sequencing as confirmatory and genotyping methods, respectively. Haplotype analyses were performed to examine associations between genetic variants and epidemiological variables. At microscopy examination, G. duodenalis was the most prevalent parasite found (29.3%, 91/311; 95% CI: 24.3-34.7), followed by Entamoeba spp. (18.7%, 58/311; 14.5-23.4), Ascaris lumbricoides (1.3%, 4/311; 0.4-3.3), and Taenia sp. (0.3%, 1/311; 0.01-1.8). qPCR confirmed the presence of G. duodenalis in 76.9% (70/91) of the microscopy-positive samples. Of them, 65.9% (60/91) were successfully genotyped. Assemblage B (68.3%, 41/60) was more prevalent than assemblage A (28.3%, 17/60). Mixed A + B infections were identified in two samples (3.3%, 2/60). These facts, together with the absence of animal-adapted assemblages, suggest that human transmission of giardiasis was primarily anthroponotic. Efforts to control G. duodenalis (and other fecal-orally transmitted pathogens) should focus on providing safe drinking water and improving sanitation and personal hygiene practices.This research was funded by the Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, grant number PI19CIII/00029.S