Editorial for the Special Issue: Diagnosis, Epidemiology and Transmission Dynamics of Cryptosporidium spp. and Giardia duodenalis

EditorialS

Editorial: Anthropogenic wildlife movements and infectious diseases: Health and conservation perspectives

EditorialS

The Metallo-β-lactamase GOB Is a Mono-Zn(II) Enzyme with a Novel Active Site

Metallo-β-lactamases (MβLs) are zinc-dependent enzymes able to hydrolyze and inactivate most β-lactam antibiotics. The large diversity of active site structures and metal content among MβLs from different sources has limited the design of a pan-MβL inhibitor. Here we report the biochemical and biophysical characterization of a novel MβL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MβLs. Contrasting all other related MβLs, GOB-18 is fully active against a broad range of β-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MβLs

Forest landscape ecology and biodiversity maintenance: application to forest resources planning in spanish rural landscapes

Spanish Forestry Act enhances the development of tools for planning and monitoring forestry uses in land units beyond local forest boundaries. This requirement implies a new approach at a higher to forest ecosystem organisation level. Landscape level approach includes knowledge of composition, structure and function of different landscape elements and land uses related to a forested matrix in a region. Nowadays there are some ongoing projects from different public and private institutions aiming to settle the methodological basis and research requirements for appropriate design and monitoring of forest landscape regions. We are reporting aims and preliminary results related to one of these projects involving two characteristic forested areas in central Spain: pine forests of Burgos‐ Soria region and different forest types located in High Tajo Valley. Main topics are: • Forest landscape and area delineation criteria. • Overlook of main digital data sources for the region related to non biotic and biotic components. • Settlement of main habitat, ecosystem and landscape typologies. • Research on main grain and landscape unit size for evaluating landscape indices. Evaluation of landscape structure in relation with some frequent disturbance sources (wild fires, floods and others). • Baseline definition for monitoring diversity related to representative groups of plant and animal species. • Settlement of habitat structure for some focal species existing in the area. • Evaluation of silviculture and management in relation with biodiversity maintenance at landscape level. Methodological routines are based on GIS geo‐databases and simulation programs that includes several digital information sources and field data related to habitat distribution, species composition, silviculture, disturbances impact,… One of the outputs of this project is a methodological guide for delineation, sustainable management and monitoring the forest landscapes in Mediterranean Spain

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

Toxoplasmosis: epidemiologic study of hospitalizated cases in Spain (2000-2021)

Artículo[ES] Introducción: La toxoplasmosis es una zoonosis de distribución mundial causada por Toxoplasma gondii. El ser humano actúa como huésped intermediario dentro de un ciclo multiespecie en el que los hospedadores definitivos son los félidos. La importancia de la infección humana se centra, principalmente, en personas inmunocomprometidas y en los casos congénitos (mujeres gestantes que han transmitido la enfermedad al feto). El objetivo fue describir la epidemiología de la hospitalización con diagnóstico toxoplasmosis en España durante el período 2000-2021.Método: Se plantea un análisis descriptivo de las hospitalizaciones en España con diagnóstico de toxoplasmosis entre los años 2000-2021, a partir de datos del Registro de Atención Sanitaria Especializada. Las variables analizadas fueron: sexo, edad y Comunidades y Ciudades Autónomas (CCAA).Resultados: durante el período 2000-2021 se produjeron 8.102 altas hospitalarias con diagnóstico toxoplasmosis, (tasa de hospitalización acumulada por 100.000 hab. TH=0,81). Esta TH ha ido disminuyendo a lo largo del período, pasando de 1,55 en 2000 hasta 0,48 en 2021. El número de ingresos fue mayor en hombres y en éstos, se ha ido produciendo un descenso más acusado. El grupo de edad con la mayor TH acumulada fue el de 35-39 años (TH=1,96). Las CCAA con mayores TH totales fueron, Ceuta, Melilla, Islas Baleares y Cataluña. Conclusiones: Las TH por toxoplasmosis han descendido progresivamente a lo largo del período 2000-2021. Este descenso es más acusado en hombres y se debe, sobre todo, a la disminución de la TH en el grupo de edad de 35-39 años. [EN] Introduction: Toxoplasmosis is a worldwide zoonotic disease caused by Toxoplasma gondii. Humans are intermediate hosts within a multispecies cycle in which felids are definitive hosts. The importance of the human infection is restricted to immunocompromised individuals and pregnant women who can transmit the disease to the fetus. The purpose was to describe toxoplasmosis hospitalization epi-demiology in Spain during 2000-2021.Methods: A descriptive analysis of hospitalization in Spain with a diagnosis of toxoplasmosis from 2000 to 2021 was performed using data from the Specialized Health Care Registry (RAE-CMBD). The variables analyzed were sex, age and Autonomous Communities (CCAA).Results: During the period 2000-2021 there were 8,102 hospital admissions with a diagnosis of to-xoplasmosis (hospitalization rate HT = 0.81). This HT decreased throughout the period, from 1.55 (2000) to 0.48 in 2021. The number of admissions was higher in men and in these, there has been a steeper decline over the period. The age group with the highest accumulated HT was 35-39 years old (HT=1.96). The CCAA with the highest total HT were Ceuta, Melilla, Balearic Islands and Catalonia. Conclusions: HT toxoplasmosis have progressively decresed throughout 2000-2021. This decrease is more pronounced in men and is due, above all, to the decrease in HT in 35-39 age group.N

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

Presence and genetic diversity of enteric protists in captive and semi-captive non-human primates in côte d'Ivoire, Sierra Leone, and Peru

Little information is currently available on the occurrence and genetic diversity of pathogenic and commensal protist species in captive and semi-captive non-human primates (NHP) resident in zoological gardens or sanctuaries in low- and medium-income countries. In this molecular-based study, we prospectively collected individual faecal samples from apparently healthy NHP at the Abidjan Zoological Garden (AZG) in Côte d'Ivoire, the Tacugama Sanctuary (TS) in Sierra Leone, and the Quistococha Zoological Garden (QZG) in Peru between November 2018 and February 2020. We evaluated for the presence of pathogenic (Cryptosporidium spp., Entamoeba histolytica, Giardia duodenalis, Blastocystis sp., Enterocytozoon bieneusi, Balantioides coli) and commensal (Entamoeba dispar, Troglodytella abrassarti) protist species using PCR methods and Sanger sequencing. Giardia duodenalis was the most prevalent species found (25.9%, 30/116), followed by Blastocystis sp. (22.4%, 26/116), and E. dispar (18.1%, 21/116). We detected E. bieneusi (4.2%, 1/24) and T. abrassarti (12.5%, 3/24) only on NHP from AZG. Cryptosporidium spp., E. histolytica, and B. coli were undetected at the three sampling sites investigated here. Sequence analyses revealed the presence of zoonotic sub-assemblages BIII (n = 1) in AZG and BIV (n = 1) in TS within G. duodenalis. We identified Blastocystis subtype ST3 (100%, 6/6) in AZG, ST1 (80.0%, 12/15), ST2 (6.7%, 1/15), and ST3 (13.3%, 2/15) in TS, and ST2 (80.0%, 4/5) and ST3 (20.0%, 1/5) in QZG. The only E. bieneusi isolate detected here was identified as zoonotic genotype CAF4. Our PCR-based data indicate that potentially pathogenic protist species including G. duodenalis, Blastocystis sp., E. bieneusi, and B. coli are present at variable rates in the three NHP populations investigated here. The identification of zoonotic genotypes within these species indicates that human-NHP transmission is possible, although the extent and directionality of these events need to be elucidated in future molecular surveys.This study was funded by the Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness under project PI16CIII/00024. David González-Barrio is the recipient of a ‘Sara Borrell’ postdoctoral fellowship (CD19CIII/00011) funded by the Spanish Ministry of Science, Innovation and Universities. Alejandro Dashti is the recipient of a PFIS contract (FI20CIII/00002) funded by the Spanish Ministry of Science and Innovation and Universities.S

Molecular Detection and Characterization of Blastocystis sp. and Enterocytozoon bieneusi in Cattle in Northern Spain

Some enteric parasites causing zoonotic diseases in livestock have been poorly studied or even neglected. This is the case in stramenopile Blastocystis sp. and the microsporidia Enterocytozoon bieneusi in Spain. This transversal molecular epidemiological survey aims to estimate the prevalence and molecular diversity of Blastocystis sp. and E. bieneusi in cattle faecal samples (n = 336) in the province of Álava, Northern Spain. Initial detection of Blastocystis and E. bieneusi was carried out by polymerase chain reaction (PCR) and Sanger sequencing of the small subunit (ssu) rRNA gene and internal transcribed spacer (ITS) region, respectively. Intra-host Blastocystis subtype diversity was further investigated by next generation amplicon sequencing (NGS) of the ssu rRNA gene in those samples that tested positive by conventional PCR. Amplicons compatible with Blastocystis sp. and E. bieneusi were observed in 32.1% (108/336, 95% CI: 27.2-37.4%) and 0.6% (2/336, 95% CI: 0.0-1.4%) of the cattle faecal samples examined, respectively. Sanger sequencing produced ambiguous/unreadable sequence data for most of the Blastocystis isolates sequenced. NGS allowed the identification of 10 Blastocystis subtypes including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26. All Blastocystis-positive isolates involved mixed infections of 2-8 STs in a total of 31 different combinations. The two E. bieneusi sequences were confirmed as potentially zoonotic genotype BEB4. Our data demonstrate that Blastocystis mixed subtype infections are extremely frequent in cattle in the study area. NGS was particularly suited to discern underrepresented subtypes or mixed subtype infections that were undetectable or unreadable by Sanger sequencing. The presence of zoonotic Blastocystis ST1, ST3, and ST5, and E. bieneusi BEB4 suggest cross-species transmission and a potential risk of human infection/colonization.This research was funded by the Health Institute Carlos III (ISCIII), Ministry of Science, Innovation and Universities (Spain), grant numbers PI16CIII/00024 and USDA-ARS Project No: 8042–32000-112–00-D.S