The effect of COVID-19 on the frequency of Pneumocystis jirovecii pneumonia: a monocentric, retrospective, and observational study.

BACKGROUND: Pneumocystis jirovecii pneumonia (PCP) and SARS-CoV2 share some similarities in their effects on the respiratory system, clinical presentation, and management. The COVID-19 pandemic required rapid action to curb transmission and mitigate its lethiferous impact. Non-pharmaceutical interventions (NPIs) were globally adopted. We hypothesized that these measures reduced the transmission and acquisition of P. jirovecii in both hospital and community settings. METHODS: We conducted a retrospective observational study on 2950 respiratory specimens from patients with suspected pulmonary infection, analyzed at the Laboratory of Parasitology Unit of the Policlinico Tor Vergata of Rome, Italy, from January 2014 to December 2022. RESULTS: We show a significant reduction in the frequency of PCP in the COVID-19 pandemic era compared to the previous period. Among the four sequence types of P. jirovecii identified, genotype 1 was the most prevalent (37%). We observed a non-significant trend of decreasing cases with genotype 1 and increasing cases with genotype 3 over the study period. CONCLUSIONS: The nationwide implementation of NPIs against COVID-19 may have changed the microbiological landscape of exposure, thereby decreasing the exposure to P. jirovecii and consequently reducing the incidence of PCP.

Pneumocystis jirovecii pneumonia in patients with previously untreated acute myeloid leukaemia.

BACKGROUND: Several studies in immunocompromised patients, such as those with HIV infection, undergoing cancer chemotherapy or organ transplant, have led to the development of guidelines on the use of prophylaxis to prevent Pneumocystis jirovecii pneumonia (PJP), in these specific conditions. Instead, since the association between PJP and acute myeloid leukaemia (AML) is not clearly defined, the role of prophylaxis in patients with AML is not yet established. METHODS: We retrospectively analysed 251 consecutive patients with newly diagnosed non-M3-AML, admitted at the Hematology Unit of University Tor Vergata in Rome, during the period 2010-2020. The aim of the study was to evaluate the incidence of PJP among AML patients during their first hospital admission, and to identify subjects at a high risk to develop PJP. RESULTS: Among 251 consecutive patients with non-M3-AML, 67 bronchoalveolar lavages (BAL) were performed. PJP was proven in 11/67 (16.7%) subjects undergoing BAL (11 males, median age 71 years), with an incidence of 4.3%. The most common reason for BAL execution were radiological findings such as ground-glass opacities (6/11, 55%) and atypical patterns like consolidations and nodules (5/11, 45%). One patient died because of PJP after 11 days of trimethoprim/sulfamethoxazole therapy. In multivariate analysis older age and smoking habit were independent factors significantly associated with PJP (p = .021 and 0.017 respectively). CONCLUSION: We conclude that PJP infection is not uncommon among patients with AML. If intensive chemotherapy is planned, physicians should be aware of this risk and prophylaxis should be considered, particularly in older patients.

Regional spread of contact lens-related Acanthamoeba keratitis in Italy.

Acanthamoeba ocular infections, known as Acanthamoeba keratitis, are an emerging problem among contact lens wearers. Infections mediated by Acanthamoeba are uncommon, but they can be underestimated due to poor awareness and delayed diagnosis. The routine use of rapid and cost-effective molecular methods like Real Time PCR for the diagnosis of this important pathogen could improve diagnosis and therapy outcome. This report describes the detection by Real Time PCR assay of six T4 and one T3 Acanthamoeba infections, as the first reported cases in Tuscany, Italy.

Update on Acanthamoeba jacobsi genotype T15, including full-length 18S rDNA molecular phylogeny.

Free-living amoebae of the genus Acanthamoeba are worldwide present in natural and artificial environments, and are also clinically important, as causative agents of diseases in humans and other animals. Acanthamoeba comprises several species, historically assigned to one of the three groups based on their cyst morphology, but presently recognized as at least 20 genotypes (T1-T20) on the basis of their nuclear 18S ribosomal RNA (rRNA) gene (18S rDNA) sequences. While strain identification may usually be achieved targeting short (<500 bp) 18S ribosomal DNA (rDNA) fragments, the use of full-length gene sequences (>2200 bp) is necessary for correct genotype description and reliable molecular phylogenetic inference. The genotype T15, corresponding to Acanthamoeba jacobsi, is the only genotype described on the basis of partial sequences (~1500 bp). While this feature does not prevent the correct identification of the strains, having only partial sequences renders the genotype T15 not completely defined and may furthermore affect its position in the Acanthamoeba molecular tree. Here, we complete this gap, by obtaining full-length 18S rDNA sequences from eight A. jacobsi strains, genotype T15. Morphologies and physiological features of isolated strains are reported. Molecular phylogeny based on full 18S rDNA confirms some previous suggestions for a genetic link between T15 and T13, T16, and T19, with T19 as sister-group to T15.

Pneumocystis jirovecii genotyping: experience in a tertiary-care hospital in Northern Italy.

Respiratory samples from Pneumocystis jirovecii pneumonia (PJP) cases collected at a tertiary-care university hospital in Modena were analyzed for the presence of specific polymorphisms in the mitochondrial large subunit ribosomal RNA (mtLSU-rRNA). Retrospectively, 57 cases were selected in a six-year period and 34 out of the 57 processed BAL samples returned PCR positive results, thus allowing further molecular analysis. The following P.jirovecii genotype distribution was observed: genotype 3 (50%), genotype 2 (23%), genotype 1 (18%), genotypes 1 or 4 (9%). These data add novel insights on P.jirovecii epidemiology, investigating a previously unstudied area of Northern Italy. A peculiar local distribution is highlighted with respect to other areas within the national panorama, thus encouraging further in-depth studies in an attempt to better understand the overall situation concerning P.jirovecii genotype circulation.

Identification and typing of free-living Acanthamoeba spp. by MALDI-TOF MS Biotyper.

Over the years, the potential pathogenicity of Acanthamoeba for humans and animals has gained increasing attention from the scientific community. More than 24 species belong to this genus, however only some of them are causative agents of keratitis and encephalitis in humans. Due to technical difficulties in diagnosis, these infections are likely to be under-detected. The introduction of 18S rDNA amplification for the identification of Acanthamoeba has dramatically enhanced diagnosis performances, but the attestation of genotyping requires supplementary sequencing-based procedures. In this study, 15 Acanthamoeba strains were collected and grown on nutrient agar media. Each strain was genotyped by end-point PCR assay for the amplification of the 18S rDNA gene and the genotype was assigned by sequencing analysis through neighbor joining phylogenetic tree. In order to optimize standardization of the MALDI-TOF MS assay, we established the collection time point at the cystic phase. Two strains of each genotype were randomly chosen to customize the biotyper database. For all strains, 24 spectral measurements were acquired and submitted to identification and cluster analysis of spectra. The obtained results highlighted the correct identification of Acanthamoeba strains and the overlapping of spectra dendrogram clusters to the 18S genotype assignations. In conclusion, the MALDI-TOF MS Biotyper revealed the capability to identify and genotype the Acanthamoeba strains, providing a new frontier in the diagnostic identification of amaebae and in taxonomic and phylogenetic studies.

Genotypic heterogeneity based on 18S-rRNA gene sequences among Acanthamoeba isolates from clinical samples in Italy.

Acanthamoeba keratitis (AK) is an ocular disease caused by members of a genus of free-living amoebae and it is associated predominantly with contact lens (CL) use. This study reports 55 cases of AK diagnosed in Italy. Genotype identification was carried out by PCR assay followed by sequence analysis of the 18S rRNA gene using the genus specific primers JDP1 and JDP2. Genotype assignment was based on phenetic analysis of the ASA.S1 subset of the small-subunit rRNA gene sequences. The material has been collected at the Polyclinic Tor Vergata of Rome for a total of 19 isolates and at the Polyclinic Hospital of Bari (36 isolates). Thirty-three out of the 55 genetically characterized isolates were assigned to the genotype T4. Ten isolates were identified as belonging to the genotype T15 thus confirming the first association between the genotype T15 and human amoebic keratitis previously described from the same area. We underline the occurrence of the genotype T3 and T11 identified for the first time in the country.

Detection and genotyping of Dientamoeba fragilis from symptomatic patients: New insights from Italy into a little-known gastrointestinal protozoan.

Dientamoeba fragilis (D. fragilis) represents a common protozoan in both high and low income countries. Despite this, epidemiological data on dientamoebiasis are still limited, and it is possible that the actual prevalence rates of D. fragilis have been underestimated due to the challenges in its detection and identification. In the present study, symptomatic patients from Rome (Central Italy) were surveyed for two years to determine D. fragilis percentage of infection and genotypes. Stool samples collection was performed over 864 patients, DNA extracted, and RT-PCR performed by the SeeGene Allplex™ Gastrointestinal Parasite Panel Assays. Seventy-nine resulted positive for D. fragilis (9.1%). Co-infections were detected in 22 isolates: 21 displayed Blastocystis sp. + D. fragilis (27.8%). Based on the sequence of a central fragment of the SSU rRNA gene, only genotype 1 was identified. These findings are among the few available data regarding genetic diversity of D. fragilis in Italy. Large-scale human and animal research are required to enhance our knowledge of prevalence, host range, genetic variability and zoonotic transmission of this little-known intestinal protozoan.

Acanthamoeba Sequence Types and Allelic Variations in Isolates from Clinical and Different Environmental Sources in Italy.

The genus Acanthamoeba comprises free-living amoebae distributed in a wide variety of environments. These amoebae are clinically significant, causing opportunistic infections in humans and other animals. Despite this, limited data on Acanthamoeba sequence types and alleles are available in Italy. In the present study, we analyzed all Acanthamoeba sequences deposited from Italy with new positive Acanthamoeba clinical samples from symptomatic AK cases, to provide an overview of the genetic variants' spatial patterns from different sources within the Italian context. A total of 137 Acanthamoeba sequences were obtained. Six sequence types were identified: T2/6, T3, T4, T11, T13, and T15. Only T4 and T15 were found in both sources. The Acanthamoeba T4 sequence type was found to be the most prevalent in all regions, accounting for 73% (100/137) of the Italian samples analyzed. The T4 sequence type demonstrated significant allelic diversity, with 30 distinct alleles from clinical and/or environmental samples. These outcomes enabled a better understanding of the distribution of Acanthamoeba isolates throughout Italy, reaffirming its well-recognized ubiquity. Acanthamoeba isolates analysis from keratitis, together with the environmental strains monitoring, might provide important information on different genotypes spreading. This might be useful to define the transmission pathways of human keratitis across different epidemiological scales.

The behavioral immune system in action: Psychological correlates of pathogen disgust sensitivity in healthcare professionals working in a COVID-19 hospital.

The behavioral immune system includes a set of proactive mechanisms that inhibit contact with pathogens in the first place. These mechanisms offer a sort of psychological and behavioral prophylaxis against infection. The aim of this study was to assess the functionality of the behavioral immune system under conditions of strong ecological validity. Our hypothesis was that the emotional and more primitive component of the behavioral immune system (i.e. pathogen disgust sensitivity) acts as a powerful predictor of fear of infection. The sample was made up of 101 healthcare professionals working in a COVID-19 hospital when vaccination was not yet available. We conducted a hierarchical regression analysis to assess the role of germ-related disgust in modulating levels of fear. After controlling for the significant effects of depressive symptoms and exposure to people with a confirmed diagnosis of COVID-19, we found that fear of infection was more intense in those healthcare workers who reported higher levels of germ-related disgust. Fear of infection was not related to perceived infectability, an individual variable informed by more rational cognitive appraisals. These findings show that, even in healthcare workers who can take advantage of their professional knowledge and acquired skills for rational appraisals, the most primitive component of the behavioral immune system still plays a major role in eliciting fear of COVID-19. It is likely that the psychological reactions elicited by the behavioral immune system promote preventive health behaviors in modern environments as well.

Giardia duodenalis in Wildlife: Exploring Genotype Diversity in Italy and across Europe.

Fragmented data are so far available on genotype diversity of G. duodenalis in wildlife in different countries in Europe, in particular, in Italy. In the present study, G. duodenalis sequences obtained from different Italian wild animals [12 porcupines (Hystrix cristata), 4 wild boars (Sus scrofa), 1 wolf (Canis lupus italicus), 6 Alpine chamois (Rupicapra rupicapra rupicapra)] were compared with those available from wild host species in Europe to add new data on the geographic distribution of Giardia assemblages/sub-assemblages and their transmission patterns among natural hosts. Thirty-eight sequences were obtained by MLG analysis (SSU-rRNA, bg, gdh, and tpi genes) and subsequently compared by phylogenetic and network analyses with those from wild species monitored in the last decades in Europe. The results revealed the presence of potentially zoonotic (A-AI, A-AII from wild boar; B from porcupine) and host-adapted (D from wolf; E, A-AIII from chamois) assemblages and sub-assemblages and represent the first report for Italian wild boar. The analysis did not find any evidence of spatial or host segregation for specific genetic variants, mostly shared between different hosts from different European countries. However, conflicting evidence was found in genotypic assignment, advocating for data improvement and new genomic approaches.

Molecular subtyping of Blastocystis sp. isolates from symptomatic patients in Italy.

Blastocystis sp. is the most common eukaryotic parasite in the intestinal tract of humans. Due to its potential impact in public health, we determined the Blastocystis sp. subtypes (STs) and their relative frequency in symptomatic patients living in or in the vicinity of two Italian cities (Rome and Sassari). A total of 34 Blastocystis sp. isolates corresponding to 26 single and 4 mixed infections were subtyped using partial small subunit ribosomal RNA gene sequencing. From this molecular approach, the ST distribution in the present Italian population was as follows: ST3 (47.1%), ST2 (20.6%), ST4 (17.7%), ST1 (8.8%), and ST7, and ST8 (2.9%). As in almost all countries worldwide, ST3 was the most common ST reinforcing the hypothesis of its human origin. Together with a previous preliminary report, a total of seven STs (with the addition of ST5) have been found in Italian symptomatic patients. The wide range of STs identified in the Italian population suggest that Blastocystis sp. infection is not associated with specific STs even if some STs (ST1-ST4) are predominant as reported in all other countries. Since most of the STs identified in Italian patients are zoonotic, our data raise crucial questions concerning the identification of animal reservoirs for Blastocystis sp. and the potential risks of transmission to humans.

PCR-based identification of thermotolerant free-living amoebae in Italian hot springs.

Several thermal areas, also used for leisure purposes, may represent suitable habitats for free-living amoebae (FLAs), but few studies have been carried out in search for these organisms. The aim of this study was to assess the presence and distribution of FLAs by culture detection and molecular identification, over a one year-round sampling of two sites in Central Italy. Two geothermal springs (Site A and Site B) were investigated for a total of 36 water samples. Four sets of primers were used to amplify FLA DNA from all cultures positive for amoebic growth at both 37 °C and 45 °C. Overall, 33 (91.6%) water samples produced PCR amplification. Eleven taxa were identified. The array of identified species varied over the sampling period, and differed between the two hot springs, Site A harbouring 11 taxa compared to 5 of site B. However, both sites were characterized by the most common species Vermamoeba vermiformis and Naegleria australiensis. Acanthamoeba genotypes T4 and T15 were found at low frequency. Differences in the composition between the two sites could reflect environmental changes in biotic and chemical/physical parameters. From a public health perspective, the detection of potentially pathogenic amoebae could unveil a potential risk for humans.

Fear of COVID-19 among Healthcare Workers: The Role of Neuroticism and Fearful Attachment.

Fear of becoming infected is an important factor of the complex suite of emotional reactions triggered by the COVID-19 pandemic. Among healthcare workers (HWs), fear of infection can put at risk their psychological well-being and occupational efficiency. The aim of this study was to analyze the role of personality (i.e., the big five traits) and adult attachment in predicting levels of fear (as measured by the FCV-19S) in 101 HWs employed in a COVID-19 university hospital. The three significant predictors retained by the stepwise regression model were age (beta = 0.26, t = 2.89, p < 0.01), emotional stability (i.e., the inverse of neuroticism) (beta = -0.26, t = -2.89, p < 0.01), and fearful attachment (beta = 0.25, t = 2.75, p < 0.01). Older HWs with higher levels of neuroticism and fearful attachment reported more intense fear of COVID-19. Our results can be useful to identify vulnerable subgroups of HWs and to implement selective programs of prevention based on counseling and psychological support.

Analysis of Human Gut Microbiota Composition Associated to the Presence of Commensal and Pathogen Microorganisms in Côte d'Ivoire.

BACKGROUND: The human gut microbiota is a microbial ecosystem contributing to the maintenance of host health with functions related to immune and metabolic aspects. Relations between microbiota and enteric pathogens in sub-Saharan Africa are scarcely investigated. The present study explored gut microbiota composition associated to the presence of common enteric pathogens and commensal microorganisms, e.g., Blastocystis and Entamoeba species, in children and adults from semi-urban and non-urban localities in Côte d'Ivoire. METHODS: Seventy-six stool samples were analyzed for microbiota composition by 16S rRDNA sequencing. The presence of adeno-, entero-, parechoviruses, bacterial and protozoal pathogens, Blastocystis, and commensal Entamoeba species, was analyzed by different molecular assays. RESULTS: Twelve individuals resulted negative for any tested microorganisms, 64 subjects were positive for one or more microorganisms. Adenovirus, enterovirus, enterotoxigenic Escherichia coli (ETEC), and Blastocystis were frequently detected. CONCLUSIONS: The bacterial composition driven by Prevotellaceae and Ruminococcaceae confirmed the biotype related to the traditional dietary and cooking practices in low-income countries. Clear separation in UniFrac distance in subjects co-harboring Entamoeba hartmanni and Blastocystis was evidenced. Alpha diversity variation in negative control group versus only Blastocystis positive suggested its possible regulatory contribution on intestinal microbiota. Pathogenic bacteria and virus did not affect the positive outcome of co-harbored Blastocystis.

Characterization of prevalence and genetic subtypes of Blastocystis sp. in wild and domestic Suidae of central Italy aided by amplicon NGS.

Blastocystis spp. is a common single-celled intestinal symbiont, comprising several genetic subtypes (ST) and transmissible by animal-to-animal, human-to-human, animal-to-human and, possibly, human-to-animal routes. This work was designed to explore the presence of Blastocystis in sympatric domestic and wild suids and their ability to carry zoonotic STs, in a condition of widespread opportunity to come in contact with the microorganism through their shared water and food resources, and other carriers. We sampled 42 and 37 stool samples from wild boars and domestic pigs, respectively. STs were first identified by PCR followed by Sanger sequencing. Sequences represented in double-band PCR products or in Sanger chromatograms displaying multiple peaks, were resolved by next generation sequencing (NGS). Twenty-six (61.9%) wild boar and 26 (70.2%) pig samples were PCR-positive, respectively. ST3, ST5 and ST15 were found in 3.8%, 38.4% and 80.8% of the positive wild boars and 11.5%, 88.5%, 11.5% of the positive pigs, respectively. ST1 was found only in pigs (3.8%). STs 5 and 15 were common in both groups of animals, but in reversed proportions, suggesting preferential colonization. We found significantly different ST distributions among wild boars and domestic pigs. This might indicate that lifestyle differences between the two populations influence their risk for contracting certain subtypes, or that ST5 and ST15 can colonize preferentially wild or domestic animals. Based on the STs described here, wild boars and domestic pigs can act as reservoirs with zoonotic potential. The ability of suids to carry zoonotic STs appears to be higher when using NGS than Sanger sequencing, and resolution of complex sequencing profiles is imperative before excluding the presence of STs of human concern.

Prevention and Control of Legionella and Pseudomonas spp. Colonization in Dental Units.

Introduction: Dental Unit Waterlines (DUWLs) have shown to be a source of Legionella infection. We report the experience of different dental healthcare settings where a risk management plan was implemented. Materials and methods: In a Hospital Odontostomatology Clinic (HOC) and three Private Dental Clinics (PDCs) housing 13 and six dental units (DUs), respectively, an assessment checklist was applied to evaluate staff compliance with guideline recommendations. DUWLs microbial parameters were investigated before and after the application of corrective actions. Results: In the HOC a poor adherence to good practices was demonstrated, whereas protocols were carefully applied in PDCs. L. pneumophila sg 2-15 was isolated in 31% (4/13) and 33% (2/6) of DUs in HOC and PDCs, respectively, mainly from handpieces (32%, 6/19) with counts >102 colony-forming units per milliliter (CFU/L), often associated with P. aeruginosa (68%, 13/19). The shock disinfection with 3% v/v hydrogen peroxide (HP) showed a limited effect, with a recolonization period of about 4 weeks. Legionella was eradicated only after 6% v/v HP shock disinfection and filters-installation, whilst P. aeruginosa after the third shock disinfection with a solution of 4% v/v HP and biodegradable surfactants. Conclusions: Our data demonstrate the presence and persistence of microbial contamination within the DUWLs, which required strict adherence to control measures and the choice of effective disinfectants.

Anthelminthic medicinal plants in veterinary ethnopharmacology: A network meta-analysis following the PRISMA-P and PROSPERO recommendations.

Medicinal plants may be effective against helminthic infestation in animals, but to date few studies have investigated the real impact of anthelminthic medicinal plants in veterinary ethnopharmacology. The aim of this study was to assess the geographical use of anthelminthic medicinal plants in livestock in European Union (EU), and to quantify the anthelminthic efficacy of medicinal plants in comparison with anthelminthic drugs. Surveys on the use of anthelminthic traditional medicinal plants in livestock in the EU were included in the qualitative synthesis. Studies that investigated the efficacy of anthelminthic traditional medicinal plants in animals, compared with negative control and/or anthelminthic drugs, were included in the quantitative synthesis (network meta-analysis). Twelve surveys (9 in Italy, 2 in Spain, 1 in Austria) reported the use of anthelminthic medicinal plants in livestock living in EU Countries. Data obtained from 256 animals and extracted from 6 studies were included in the network meta-analysis. Medicinal plants and drugs were more effective than negative control (standardized mean difference [SMD]: -0.60 95%CrI -0.88 to -0.31, -0.73 95%CrI -1.08 to -0.38, respectively, P < 0.001). Overall, no difference was detected between anthelminthic medicinal plants and anthelminthic drugs, namely albendazole, ivermectin, fenbendazole, and doramectin (SMD: 0.26 95%CrI -0.02 to 0.55, P > 0.05). The most effective anthelminthic medicinal plants were Artemisia absintihium, Allium sativum, and Duranta erecta. There is the strong medical need of performing adequately powered randomized controlled trials in different livestock species aimed to improve the quality of the current evidence concerning the anthelminthic efficacy of medicinal plants compared to that of the currently available antiparasitic drugs.

Immunoprophylaxis pharmacotherapy against canine leishmaniosis: A systematic review and meta-analysis on the efficacy of vaccines approved in European Union.

Leishmania (L.) infantum is a vector-borne parasite currently endemic in several Southern countries of European Union (EU), and dogs represent the main reservoir and hosts. Data from clinical trials are inconsistent with respect to the efficacy of vaccination against L. infantum infection. Therefore, a quantitative synthesis via pairwise meta-analysis was performed in agreement with the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) to increase the strength of evidence and assess the real efficacy profile of vaccines against L. infantum currently approved in EU. Data obtained from 1,394 dogs were extracted from 10 studies. The overall analysis indicated that vaccination is significantly effective in protecting against L. infantum infection (RR 0.40, 95%CI 0.23-0.72; I2 70%; P < 0.01 vs. negative controls). The subset analysis performed by excluding the effect modifiers and by considering only the studies that assessed the efficacy of vaccines currently available in EU, indicated that CaniLeish® (RR 0.38, 95%CI 0.20-0.72; I2 0%), but not Letifend® (RR 0.43, 95%CI 0.15-1.22; I2 37%), significantly protected against L. infantum infection when compared to negative controls (P < 0.05). The number needed to treat analysis showed that 3.77 (95%CI 2.59-6.94) and 10.99 (95%CI 8.28-16.34) dogs had to be treated with CaniLeish® and Letifend®, respectively, to prevent one case of infection compared to negative controls. Vaccination is effective in protecting against the risk L. infantum infection, but further studies are needed to assess whether CaniLeish® and Letifend® are characterized by similar efficacy profile.

Giardia and Cryptosporidium in inflowing water and harvested shellfish in a lagoon in Southern Italy.

Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for humans and animals, and have been found to contaminate water as well as edible shellfish all over the world. This is the first study to simultaneously investigate the presence of Giardia and Cryptosporidium in inflowing water and harvested shellfish in a geographically closed environment (Varano Lagoon, Southern Italy). Samples of treated wastewater were collected each month - at the outlet from the treatment plant, and downstream at the inlet into the lagoon - from the channels flowing into the Lagoon, together with specimens of Ruditapes decussatus and Mytilus galloprovincialis from shellfish-farms on the same lagoon. Giardia cysts were found by immunofluorescence (IF) microscopy in 16 out of 21 samples of treated wastewater and in 7 out of 21 samples from downstream water channels, and viable cysts were also detected by a beta-giardin RT-PCR. G. duodenalis Assemblages A and B were identified by small ribosomal subunit (18S-rDNA) and triosephosphate isomerase (tpi)-PCR, followed by sequencing. Cryptosporidium oocysts were found by IF in 5 out of 21 wastewater samples, and in 8 out of 21 samples from water channels. Molecular analysis identified the zoonotic species Cryptosporidium parvum by oocyst wall protein (COWP)-PCR and sequencing. Higher concentrations of Giardia cysts than Cryptosporidium oocysts were registered in almost all wastewater and water samples. IF and molecular testing of shellfish gave negative results for both protozoa. Wastewaters carrying Giardia and Cryptosporidium (oo)cysts are discharged into the Lagoon; however, the shellfish harvested in the same environment were found to be unaffected, thus suggesting that physical, ecological and climatic conditions may prevent contamination of harvested shellfish.