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Summary: Background: For Candida infections antifungal therapy is often empirical and mainly depends on locally antifungal surveillance data, which differs between geographic regions. Aims: To monitor the epidemiology and antifungal susceptibility of Candida spp. from combined axillar-groin samples in intensive care unit (ICU) patients on admission (day1, D1), day 5 (D5) and day 8 (D8). Methods: From 2020 to 2022, 675 patients from three ICUs were enrolled. Candida isolates were identified by MALDI-TOF MS and PCR. In vitro antifungals susceptibility tests (AFST) were performed for fluconazole, voriconazole, amphotericin B and anidulafungin, by concentration gradient Etest® strip technique. Results: Out of 988 swabs, 355 isolates were identified as Candida species from 232 patients, being 89 isolates retrieved from patients that remained colonised at D5 and D8. AFST was conducted for all Candida isolates. The overall rate of resistance to fluconazole was 2.7%, with 3 out of 133 C. albicans, 2 out of 89 C. parapsilosis and 2 out of 24 C. glabrata isolates identified as resistant. Voriconazole susceptibility was observed in 99.2% of the isolates, with only one C. albicans isolate identified as resistant to this triazole. All isolates were susceptible to amphotericin B and 98.5% to anidulafungin. Three Candida spp. exhibited resistance to anidulafungin, C. albicans, C. tropicalis, and C. parapsilosis. Conclusions: This study highlights the importance of C. albicans as a frequent coloniser and showed that antifungal resistance remains uncommon among Candida isolates from ICUs in Portugal. The results may contribute to better management within institutions to guide therapeutic decision making.

IntroductionCandida auris is an emerging pathogen that represents a worldwide health problem due to its global expansion, multidrug resistance, and difficult laboratory identification. Among the risk factors for colonization/infection by C. auris, a stay in an intensive care unit (ICU) stands out. This prospective multicenter study aimed to monitor the trend of the local epidemiology of Candida spp. and unveil the prevalence of C. auris.MethodsFrom 2020 to 2022, axillar/inguinal swabs were collected from adult patients at three points: upon admission (D1) and on the fifth (D5) and eighth (D8) days of their ICU stay. We employed culture-based screening methods combined with molecular techniques to identify Candida spp. down to the species level. Specific screening for Candida auris was conducted using a real-time PCR assay in combination with an improved selective culture medium, mannitol salt agar auris (MSAA). To validate the effectiveness of MSAA, a collection of reference C. auris strains representing the four major geographical clades was used.ResultsWe enrolled 675 patients, and 355 Candida isolates were retrieved from the 988 swab samples collected. From those, 185/355 (52.1%) were identified as C. albicans and 170/355 (47.9%) as non-albicans Candida (NAC). MSAA medium showed a specificity of 94.8%, albeit C. auris was not detected in this cohort. The dynamics of Candida spp. colonization by ICU were significant at the three collection points. Upon admission, C. albicans was associated with the Beatriz Ângelo Hospital ICU (p=0.003) and C. tropicalis with the general Hospital Professor Doutor Fernando Fonseca (FFH) ICU (p=0.006). C. parapsilosis and C. lusitaniae were associated with FFH ICUs, with the general ICU at D5 (p=0.047) and surgical ICU at D8 (p=0.012). The dynamics of NAC colonization by ICU were significantly different at D1 (p=0.011), D5 (p=0.047), and D8 (p=0.012).ConclusionWe developed and implemented a screening protocol for C. auris while uncovering the colonization patterns of Candida in the ICU. Our findings contribute to the optimization of overall patient management, ensuring that ICU protocols are resilient and adaptive to emerging fungal threats.

Lophomonas are flagellated protozoa that have been increasingly associated with upper and lower airway infection in humans. The prevalence and characterization of this disease in the critically ill remains poorly understood. We present a series of eleven ICU patients with confirmed Lophomonas spp. identification in respiratory samples.

Post COVID-19 condition (PCC) is defined as ongoing symptoms at ≥1 month after acute COVID-19. We investigated the risk of PCC in an international cohort according to viral variants. We included 7699 hospitalized patients in six centers (January 2020–June 2023); a subset of participants with ≥1 visit over the year after clinical recovery were analyzed. Variants were observed or estimated using Global Data Science Initiative (GISAID) data. Because patients returning for a post COVID-19 visit may have a higher PCC risk, and because the variant could be associated with the probability of returning, we used weighted logistic regressions. We estimated the proportion of the effect of wild-type (WT) virus vs. Omicron on PCC, which was mediated by Intensive Care Unit (ICU) admission, through a mediation analysis. In total, 1317 patients returned for a post COVID visit at a median of 2.6 (IQR 1.84–3.97) months after clinical recovery. WT was present in 69.6% of participants, followed by the Alpha (14.4%), Delta (8.9%), Gamma (3.9%) and Omicron strains (3.3%). Among patients with PCC, the most common manifestations were fatigue (51.7%), brain fog (32.7%) and respiratory symptoms (37.2%). Omicron vs. WT was associated with a reduced risk of PCC and PCC clusters; conversely, we observed a higher risk with the Delta and Alpha variants vs. WT. In total, 42% of the WT effect vs. Omicron on PCC risk appeared to be mediated by ICU admission. A reduced PCC risk was observed after Omicron infection, suggesting a possible reduction in the PCC burden over time. A non-negligible proportion of the variant effect on PCC risk seems mediated by increased disease severity during the acute disease.

Abstract Immunobiography describes the life-long effects of exogenous or endogenous stimuli on remodeling of immune cell biology, including the development of memory T and B-cells. The present study aimed at investigating the rhythms of changes in phenotypic features of memory T and B-cells along childhood and adolescence. A descriptive-observational investigation was conducted including 812 healthy volunteers, clustered into six consecutive age groups (9Mths–1Yr; 2Yrs; 3–4Yrs; 5–7Yrs; 8–10Yrs; 11–18Yrs). Immunophenotypic analysis of memory T-cell (CD4+ and CD8+) and B-cell subsets were performed by flow cytometry. The results pointed out that memory-related biomarkers of T and B-cells displayed a bimodal profile along healthy childhood and adolescence, regardless of sex. The first stage of changes occurs around 2Yrs, with predominance of naive cells, while the second and more prominent wave occurs around the age 8–10Yrs, with the prevalence of memory phenotypes. The neighborhood connectivity profile analysis demonstrated that the number of correlations reaches a peak at 11–18Yrs and lower values along the childhood. Males presented higher and conserved number of correlations when compared to females. Altogether, our results provide new insights into immunobiography and a better understanding of interactions among the cellular subsets studied here during childhood and adolescence.

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), can lead to hospitalisation, particularly in elderly, immunocompromised, and non-vaccinated or partially vaccinated individuals. Although vaccination provides protection, the duration of this protection wanes over time. Additional doses can restore immunity, but the influence of viral variants, specific sequences, and vaccine-induced immune responses on disease severity remains unclear. Moreover, the efficacy of therapeutic interventions during hospitalisation requires further investigation. The study aims to analyse the clinical course of COVID-19 in hospitalised patients, taking into account SARS-CoV-2 variants, viral sequences, and the impact of different vaccines. The primary outcome is all-cause in-hospital mortality, while secondary outcomes include admission to intensive care unit and length of stay, duration of hospitalisation, and the level of respiratory support required. Methods This ongoing multicentre study observes hospitalised adult patients with confirmed SARS-CoV-2 infection, utilising a combination of retrospective and prospective data collection. It aims to gather clinical and laboratory variables from around 35,000 patients, with potential for a larger sample size. Data analysis will involve biostatistical and machine-learning techniques. Selected patients will provide biological material. The study started on October 14, 2021 and is scheduled to end on October 13, 2026. Discussion The analysis of a large sample of retrospective and prospective data about the acute phase of SARS CoV-2 infection in hospitalised patients, viral variants and vaccination in several European and non-European countries will help us to better understand risk factors for disease severity and the interplay between SARS CoV-2 variants, immune responses and vaccine efficacy. The main strengths of this study are the large sample size, the long study duration covering different waves of COVID-19 and the collection of biological samples that allows future research. Trial registration The trial has been registered on ClinicalTrials.gov. The unique identifier assigned to this trial is NCT05463380.

Background: COVID-19 has become a major public health problem after the outbreak caused by SARS-CoV-2 virus. Great efforts to contain COVID-19 transmission have been applied worldwide. In this context, accurate and fast diagnosis is essential. Methods: In this prospective study, we evaluated the clinical performance of three different RNA-based molecular tests – RT-qPCR (Charité protocol), RT-qPCR (CDC (USA) protocol) and RT-LAMP – and one rapid test for detecting anti-SARS-CoV-2 IgM and IgG antibodies. Results: Our results demonstrate that RT-qPCR using the CDC (USA) protocol is the most accurate diagnostic test among those evaluated, while oro-nasopharyngeal swabs are the most appropriate biological sample. RT-LAMP was the RNA-based molecular test with lowest sensitivity while the serological test presented the lowest sensitivity among all evaluated tests, indicating that the latter test is not a good predictor of disease in the first days after symptoms onset. Additionally, we observed higher viral load in individuals who reported more than 3 symptoms at the baseline. Nevertheless, viral load had not impacted the probability of testing positive for SARS-CoV-2. Conclusion: Our data indicates that RT-qPCR using the CDC (USA) protocol in oro-nasopharyngeal swabs samples should be the method of choice to diagnosis COVID-19.

The World Health Organization (WHO) recognizes schistosomiasis as one of the Neglected Tropical Diseases targeted for global elimination in the 2030 Agenda of the Sustainable Development Goals. In Brazil, schistosomiasis mansoni is considered a public health problem, particularly prevalent among vulnerable populations living in areas with poor environmental and sanitary conditions. In 2022, the WHO published a Guideline encompassing recommendations to assist national programs in endemic countries in achieving morbidity control, eliminating schistosomiasis as a public health problem, and advancing towards interrupting transmission. The perspectives presented here, collectively prepared by members of the Oswaldo Cruz Foundation’s (Fiocruz) Schistosomiasis Translational Program (FioSchisto), along with invited experts, examine the feasibility of the WHO recommendations for the Brazilian settings, providing appropriate recommendations for public health policies applicable to the epidemiological reality of Brazil, and suggests future research to address relevant issues. In Brazil, the provision of safe water and sanitation should be the key action to achieve schistosomiasis elimination goals. The agencies involved in measures implementation should act together with the Primary Care teams for planning, executing, monitoring, and evaluating actions in priority municipalities based on their epidemiological indicators. Host snails control should prioritize judicious ecological interventions at breeding sites. The Information, Education, and Communication (IEC) strategy should be associated with water and sanitation and other control actions, actively involving school community. To identify infected carriers, FioSchisto recommends a two-stage approach of immunological and molecular tests to verify transmission interruption during the intervention and beyond. Praziquantel administration should be done under medical supervision at the Primary Care level. MDA should be considered in exceptional settings, as a measure of initial attack strategy in locations presenting high endemicity, always integrated with water and sanitation, IEC, and snail control. To assist decision-making, as well as the monitoring and evaluation of strategic actions, there is a need for an Information System. FioSchisto considers this systematization essential to make investments in strategic research to support the improvement of schistosomiasis control actions. Efforts toward schistosomiasis elimination in Brazil will succeed with a paradigm shift from the vertical prescriptive framework to a community-centered approach involving intersectoral and interdisciplinary collaboration.

The skin mycobiota plays a significant role in infection risk, pathogen transmission, and personalized medicine approaches in intensive care settings. This prospective multicenter study aimed to enhance our understanding of intensive care units’ (ICUs’) Candida colonization dynamics, identify modifiable risk factors, and assess their impact on survival risk. Specimens were taken from 675, 203, and 110 patients at the admission (D1), 5th (D5), and 8th (D8) days of ICU stay, respectively. The patient’s demographic and clinical data were collected. Candida isolates were identified by conventional culture-based microbiology combined with molecular approaches. Overall, colonization was 184/675 (27.3%), 87/203 (42.8%), and 58/110 (52.7%) on D1, D5, and D8, respectively. Candida colonization dynamics were significantly associated with ICU type (odds ratio (OR) = 2.03, 95% CI 1.22–3.39, p = 0.007), respiratory infection (OR = 1.74, 95% CI 1.17–2.58, p = 0.006), hemodialysis (OR = 2.19, 95% CI 1.17–4.10, p = 0.014), COVID-19 (OR = 0.37, 95% CI 0.14–0.99, p = 0.048), and with a poor 3-month outcome (p = 0.008). Skin Candida spp. colonization can be an early warning tool to generate valuable insights into the epidemiology, risk factors, and survival rates of critically ill patients, and should be considered for epidemiological surveillance.

IntroductionThe pandemic caused by SARS-CoV-2 has had a major impact on health systems. Vaccines have been shown to be effective in improving the clinical outcome of COVID-19, but they are not able to fully prevent infection and reinfection, especially that caused by new variants.MethodsHere, we tracked for 450 days the humoral immune response and reinfection in 52 healthcare workers from Brazil. Infection and reinfection were confirmed by RT-qPCR, while IgM and IgG antibody levels were monitored by rapid test.ResultsOf the 52 participants, 19 (36%) got reinfected during the follow-up period, all presenting mild symptoms. For all participants, IgM levels dropped sharply, with over 47% of them becoming seronegative by the 60th day. For IgG, 90% of the participants became seropositive within the first 30 days of follow-up. IgG antibodies also dropped after this period reaching the lowest level on day 270 (68.5 ± 72.3, p

BackgroundSchistosomiasis is a parasitic disease associated with poverty. It is estimated that 7.1 million people are infected with Schistosoma mansoni in Latin America, with 95% of them living in Brazil. Accurate diagnosis and timely treatment are important measures to control and eliminate schistosomiasis, but diagnostic improvements are needed to detect infections, especially in areas of low endemicity.MethodologyThis research aimed to evaluate the performance of 11 diagnostic tests using latent class analysis (LCA). A cross-sectional survey was undertaken in a low endemicity area of the municipality of Malacacheta, Minas Gerais, Brazil. Feces, urine, and blood samples were collected from 400 residents older than 6 years of age, who had not been treated with praziquantel in the 12 months previous to the collection of their samples. The collected samples were examined using parasitological (Helm Test® kit Kato-Katz), nucleic acid amplification tests -NAATs (PCR, qPCR and LAMP on urine; PCR-ELISA, qPCR and LAMP on stool), and immunological (POC-CCA, the commercial anti-Schistosoma mansoni IgG ELISA kit from Euroimmun, and two in-house ELISA assays using either the recombinant antigen PPE or the synthetic peptide Smp150390.1) tests.ResultsThe positivity rate of the 11 tests evaluated ranged from 5% (qPCR on urine) to 40.8% (commercial ELISA kit). The estimated prevalence of schistosomiasis was 12% (95% CI: 9–15%) according to the LCA. Among all tests assessed, the commercial ELISA kit had the highest estimated sensitivity (100%), while the Kato-Katz had the highest estimated specificity (99%). Based on the accuracy measures observed, we proposed three 2-step diagnostic approaches for the active search of infected people in endemic settings. The approaches proposed consist of combinations of commercial ELISA kit and NAATs tests performed on stool. All the approaches had higher sensitivity and specificity than the mean values observed for the 11 tests (70.4 and 89.5%, respectively).ConclusionWe showed that it is possible to achieve high specificity and sensitivity rates with lower costs by combining serological and NAATs tests, which would assist in the decision-making process for appropriate allocation of public funding aiming to achieve the WHO target of eliminating schistosomiasis as a public health problem by 2030.

ABSTRACT: The failure of artemisinin combination therapy (ACT) in malaria patients returning from endemic regions may be driven by parasite resistance to this treatment. ACT is used globally as the first-line treatment for Plasmodium falciparum malaria. However, artemisinin-resistant strains of P. falciparum have emerged and spread across Southeast Asia, with the risk of reaching high malaria burden regions in Africa and elsewhere. Here, we report on two malaria imported cases from Africa with possible parasite resistance to the ACT artemether-lumefantrine (AL).Case presentation: Two middle-aged males returning from Angola and Mozambique developed malaria symptoms in Portugal, where they were diagnosed and received treatment with AL as hospital inpatients. After apparent cure and discharge from hospital, these individuals returned to hospital showing signs of late clinical failure. Molecular analysis was performed across a number of drug resistance associated genes. No evidence of pfk13-mediated artemisinin resistance was found. Both subjects had complete parasite clearance after treatment with non-ACT antimalarials.Conclusion: Our case-studies highlights the need for close monitoring of signs of unsatisfactory antimalarial efficacy among AL treated patients and the possible implication of other genes or mutations in the parasite response to ACTs.

BackgroundAccurate diagnosis followed by timely treatment is an effective strategy for the prevention of complications together with reducing schistosomiasis transmission. Recombinase Polymerase Amplification (RPA) is a simple, rapid, sensitive, and specific isothermal method with low resource needs. This research aimed at the development and optimisation of a real-time (RT) and a lateral flow (LF) RPA assay for the detection of Schistosoma mansoni.MethodologyRecombinase Polymerase Amplification reactions were performed at full- (as recommended) and half-volumes (to reduce costs), with RT or LF detection systems targeting the S. mansoni mitochondrial minisatellite region. The specificity was assessed using gDNA from other Schistosoma species, helminths co-endemic with S. mansoni, human stool, and urine, and Biomphalaria snail hosts. The analytical sensitivity was evaluated using serial dilutions of gDNA, synthetic copies of the target, and single eggs. The ability of both assays to detect the S. mansoni DNA in human urine and stool samples was also tested. The long-term stability of the RT-RPA reagents was evaluated by storing the reaction components in different temperature conditions for up to 3 weeks.ResultsThe RT- and the LF-RPA (SmMIT- and SmMIT-LF-RPA, respectively) presented similar results when used full- and half-volumes, thus the latter was followed in all experiments. The SmMIT-RPA was 100% specific to S. mansoni, able to detect a single egg, with a limit of detection (LOD) of down to 1 fg of gDNA and one synthetic copy of the target. The assay was able to detect S. mansoni DNA from stool containing 1 egg/g and in spiked urine at a concentration of 10 fg/μl. SmMIT-RPA reagents were stable for up to 3 weeks when kept at 19°C, and 2 weeks when stored at 27°C. The SmMIT-LF-RPA cross-reacted with Clinostomidae, presented the LOD of 10 fg and one synthetic copy of the target, being able to detect a single egg and 1 egg/g in a stool sample. The LOD in spiked urine samples was 10 pg/μl.ConclusionThe half-volume SmMIT-RPA is a promising method to be used in the field. It is specific, sensitive, robust, and tolerant to inhibitors, with a long-term stability of the reaction components and the real-time visualisation of results.

Abstract Background Schistosomiasis a neglected tropical disease endemic in Brazil. It is caused by the trematode Schistosoma mansoni, which is transmitted by snails of the genus Biomphalaria. Among measures used to control and eliminate schistosomiasis, accurate mapping and monitoring of snail breeding sites are recommended. Despite the limitations of parasitological methods, they are still used to identify infected snails. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cost-effective diagnostic method for the identification of infected snails. In the work reported here, we aimed to validate the use of LAMP for the detection of S. mansoni in snails of the genus Biomphalaria. Methods Snails were collected in five municipalities of the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil. Snails were pooled according to collection site and then squeezed for the detection of S. mansoni and other trematode larvae. Pooled snails were subjected to pepsin digestion and DNA extraction. Molecular assays were performed for species-specific identification and characterization of the samples. A previously described LAMP assay was adapted, evaluated, and validated using laboratory and field samples. Results Using the parasitological method described here, S. mansoni cercariae were detected in snails from two collection sites, and cercariae of the family Spirorchiidae were found in snails from one site. The snails were identified by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP). Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites. Biomphalaria kuhniana, which is resistant to S. mansoni infection, was found in the remaining sites. Multiplex, low stringency (LS), and conventional PCR allowed the detection of positive snails in four additional sites. Trematodes belonging to the families Strigeidae and Echinostomatidae were detected by multiplex PCR in two sites. The LAMP assay was effective in detecting the presence of S. mansoni infection in laboratory (7 days post-infection) and field samples with no cross-reactivity for other trematodes. When compared to LS and conventional PCR, LAMP showed 100% specificity, 85.7% sensitivity, and a κ index of 0.88. Conclusions Our findings suggest that LAMP is a good alternative method for the detection and monitoring of transmission foci of S. mansoni, as it was three times as effective as the parasitological examination used here for the detection of infection, and is more directly applicable in the field than other molecular techniques. Graphical abstract

Yeast species other than Candida albicans are becoming more clinically relevant in the Intensive Care Unit (ICU) landscape, making it important to identify them correctly. The aim of this study was to evaluate the concordance of the identification of clinical isolates from ICUs, at the species level, by conventional and molecular methods. All isolates (n = 371) underwent identification by cultural, MALDI-TOF MS and PCR. The direct concordance between conventional and molecular identification was 92% (341/371). These results allow us to conclude that culture-based methodologies are still useful to reliably identify the most frequent yeasts, but for rare, uncommon or cryptic species, technologies such as MALDI-TOF MS or PCR are needed.

The frequency of Candida spp. isolates as a cause of hospital infections has risen, and in vitro antifungal susceptibility testing (AST) plays an increasingly important role in guiding therapeutic decisions. This multicenter study aimed to determine AST pattern of Candida spp. isolates from Intensive Care Unit (ICU) patients. In total, 674 patients were enrolled, and axillar/inguinal swabs were collected at admission and during the ICU stay (5th and 8th day). In vitro AST was performed on 355 Candida spp. isolates, according to the concentration gradient Etest® strip technique. The overall susceptibility rates were 100%, 99.7%, 98.3%, and 97.7% for amphotericin B, voriconazole, anidulafungin, and fluconazole, respectively. The current study demonstrates that antifungal resistance remains infrequent among Candida spp. isolates in Portugal’s ICUs.

Staphylococcus saprophyticus is a primary cause of community-acquired urinary tract infections (UTIs) in young women. S. saprophyticus colonizes humans and animals but basic features of its molecular epidemiology are undetermined. We conducted a phylogenomic analysis of 321 S. saprophyticus isolates collected from human UTIs worldwide during 1997–2017 and 232 isolates from human UTIs and the pig-processing chain in a confined region during 2016–2017. We found epidemiologic and genomic evidence that the meat-production chain is a major source of S. saprophyticus causing human UTIs; human microbiota is another possible origin. Pathogenic S. saprophyticus belonged to 2 lineages with distinctive genetic features that are globally and locally disseminated. Pangenome-wide approaches identified a strong association between pathogenicity and antimicrobial resistance, phages, platelet binding proteins, and an increased recombination rate. Our study provides insight into the origin, transmission, and population structure of pathogenic S. saprophyticus and identifies putative new virulence factors.

The tegument of Schistosoma mansoni is involved in essential functions for parasite survival and is known to stimulate immune responses in pre-clinical vaccine trials. Smtal-9, a member of the tegument-allergen-like (TAL) family, is one of the components of the tegument of schistosomula recognized by sera from immunized and protected mice. In this work, we assessed the role of Smtal-9 in parasite survival using the RNAi approach. Also, we cloned and expressed a recombinant form of Smtal-9 and evaluated its ability to induce protection in mice. Smtal-9 knockdown did not impact parasite survival in vitro, but significantly decreased schistosomula size. Additionally, significant reduction in both parasite and egg burdens were observed in mice inoculated with Smtal-9-knockdown schistosomula. Immunization using the Smtal-9 as an antigen conferred partial protection against challenge infection. Overall, our results indicate that Smtal-9 is a candidate target for drug and/or vaccine development due to its important role in parasite biology and survival.

Background: Aspergillus fumigatus is a saprophytic fungus, ubiquitous in the environment and responsible for causing infections, some of them severe invasive infections. The high morbidity and mortality, together with the increasing burden of triazole-resistant isolates and the emergence of new risk groups, namely COVID-19 patients, have raised a crescent awareness of the need to better comprehend the dynamics of this fungus. The understanding of the epidemiology of this fungus, especially of CAPA isolates, allows a better understanding of the interactions of the fungus in the environment and the human body. Methods: In the present study, the M3 markers of the STRAf assay were used as a robust typing technique to understand the connection between CAPA isolates and isolates from different sources (environmental and clinical-human and animal). Results: Of 100 viable isolates that were analyzed, 85 genotypes were found, 77 of which were unique. Some isolates from different sources presented the same genotype. Microsatellite genotypes obtained from A. fumigatus isolates from COVID+ patients were all unique, not being found in any other isolates of the present study or even in other isolates deposited in a worldwide database; these same isolates were heterogeneously distributed among the other isolates. Conclusions: Isolates from CAPA patients revealed high heterogeneity of multi-locus genotypes. A genotype more commonly associated with COVID-19 infections does not appear to exist.

Biofilm formation has been shown to be critical to the success of uropathogens. Although Staphylococcus saprophyticus is a common cause of urinary tract infections, its biofilm production capacity, composition, genetic basis, and origin are poorly understood. We investigated biofilm formation in a large and diverse collection of S. saprophyticus (n = 422). Biofilm matrix composition was assessed in representative strains (n = 63) belonging to two main S. saprophyticus lineages (G and S) recovered from human infection, colonization, and food-related environment using biofilm detachment approach. To identify factors that could be associated with biofilm formation and structure variation, we used a pangenome-wide association study approach. Almost all the isolates (91%; n = 384/422) produced biofilm. Among the 63 representative strains, we identified eight biofilm matrix phenotypes, but the most common were composed of protein or protein–extracellular DNA (eDNA)–polysaccharides (38%, 24/63 each). Biofilms containing protein–eDNA–polysaccharides were linked to lineage G and environmental isolates, whereas protein-based biofilms were produced by lineage S and infection isolates (p < 0.05). Putative biofilm-associated genes, namely, aas, atl, ebpS, uafA, sasF, sasD, sdrH, splE, sdrE, sdrC, sraP, and ica genes, were found with different frequencies (3–100%), but there was no correlation between their presence and biofilm production or matrix types. Notably, icaC_1 was ubiquitous in the collection, while icaR was lineage G-associated, and only four strains carried a complete ica gene cluster (icaADBCR) except one that was without icaR. We provided evidence, using a comparative genomic approach, that the complete icaADBCR cluster was acquired multiple times by S. saprophyticus and originated from other coagulase-negative staphylococci. Overall, the composition of S. saprophyticus biofilms was distinct in environmental and clinical isolates, suggesting that modulation of biofilm structure could be a key step in the pathogenicity of these bacteria. Moreover, biofilm production in S. saprophyticus is ica-independent, and the complete icaADBCR was acquired from other staphylococci.

Schistosomiasis remains one of the most important neglected tropical diseases in the world. It mainly affects developing countries, where it often coexists with malnutrition. Despite this, few studies have investigated the relationship between schistosomiasis and malnutrition. Herein, we evaluate the impact of malnutrition on experimental S. mansoni infection. Mice were divided into 5 groups: Control (Ctrl) diet (14% protein and 10% lipids), low-protein 3% (LP 3%), low-protein 8% (LP 8%), low-fat 2.5% (LF 2.5%), and low-fat 5% (LF 5%). Mice were fed with their respective diets and were infected when a difference of approximately 20% in the body weight between mice from any experimental group and mice from the control group was achieved. Nutritional, parasitological, and immunological parameters were assessed either just before infection and/or approximately 50 days later before mice were perfused. Our results showed that the 3% low-protein diet was the only one capable of establishing malnutrition in mice. Mice fed with this diet showed: (i) significant reduction in body weight and serum albumin levels before infection, (ii) decreased levels of all biochemical parameters evaluated before perfusion, (iii) decreased numbers of schistosome eggs trapped in intestines and impaired parasite fecundity, (iv) a delay in the granuloma development with a smaller granuloma area, and (v) reduced levels of IL-4 and IFN-γ in the liver. Our findings demonstrate that low protein supply leads to malnutrition in mice and impacts the cytokine milieu in the liver and granuloma formation. Additionally, the establishment of our murine malnutrition model will enable future studies aiming to better understand the complex relationships between nutrition, immune responses, and infection outcome.

Invasive fungal infections (IFI) have significantly increased over the past years due to advances in medical care for the at-risk immunocompromised population. IFI are often difficult to diagnose and manage, and can be associated with substantial morbidity and mortality. This study aims to contribute to understanding the etiology of invasive and subcutaneous fungal infections, their associated risk factors, and to perceive the outcome of patients who developed invasive disease, raising awareness of these infections at a local level but also in a global context. A laboratory surveillance approach was conducted over a seven-year period and included: (i) cases of invasive and subcutaneous fungal infections caused by filamentous/dimorphic fungi, confirmed by either microscopy or positive culture from sterile samples, (ii) cases diagnosed as probable IFI according to the criteria established by EORTC/MSG when duly substantiated. Fourteen Portuguese laboratories were enrolled. Cases included in this study were classified according to the new consensus definitions of invasive fungal diseases (IFD) published in 2020 as follows: proven IFI (N = 31), subcutaneous fungal infection (N = 23). Those proven deep fungal infections (N = 54) totalized 71.1% of the total cases, whereas 28.9% were classified as probable IFI (N = 22). It was possible to identify the etiological fungal agent in 73 cases (96%). Aspergillus was the most frequent genera detected, but endemic dimorphic fungi represented 14.47% (N = 11) of the total cases. Despite the small number of cases, a high diversity of species were involved in deep fungal infections. This fact has implications for clinical and laboratory diagnosis, and on the therapeutic management of these infections, since different species, even within the same genus, can present diverse patterns of susceptibility to antifungals.

Invasive pulmonary aspergillosis (IPA) has become a recognizable complication in coronavirus disease 2019 (COVID-19) patients admitted to intensive care units (ICUs). Alveolar damage in the context of acute respiratory distress syndrome (ARDS) appears to be the culprit in facilitating fungal invasion in COVID-19 patients, leading to a COVID-19-associated pulmonary aspergillosis (CAPA) phenomenon. From November 2020 to 15 February 2021, 248 COVID-19 patients were admitted to our ICUs, of whom ten patients (4% incidence) were classified as either probable (six) or possible (four) CAPA cases. Seven patients had positive cultural results: Aspergillus fumigatus sensu stricto (five), A. terreus sensu stricto (one), and A. welwitschiae (one). Five patients had positive bronchoalveolar lavage (BAL) and galactomannan (GM), and two patients had both positive cultural and GM criteria. All but two patients received voriconazole. Mortality rate was 30%. Strict interpretation of classic IPA definition would have resulted in eight overlooked CAPA cases. Broader diagnostic criteria are essential in this context, even though differentiation between Aspergillus colonization and invasive disease might be more challenging. Herein, we aim to raise awareness of CAPA in view of its potential detrimental outcome, emphasizing the relevance of a low threshold for screening and early antifungal treatment in ARDS patients.

Malaria is one of the ‘big three’ killer infectious diseases, alongside tuberculosis and HIV. In non-endemic areas, malaria may occur in travelers who have recently been to or visited endemic regions. The number of imported malaria cases in Portugal has increased in recent years, mostly due to the close relationship with the community of Portuguese language countries. Samples were collected from malaria-infected patients attending Centro Hospitalar Lisboa Ocidental (CHLO) or the outpatient clinic of Instituto de Higiene e Medicina Tropical (IHMT-NOVA) between March 2014 and May 2021. Molecular characterization of Plasmodium falciparum pfk13 and pfmdr1 genes was performed. We analyzed 232 imported malaria cases. The majority (68.53%) of the patients came from Angola and only three patients travelled to a non-African country; one to Brazil and two to Indonesia. P. falciparum was diagnosed in 81.47% of the cases, P. malariae in 7.33%, P. ovale 6.47% and 1.72% carried P. vivax. No mutations were detected in pfk13. Regarding pfmdr1, the wild-type haplotype (N86/Y184/D1246) was also the most prevalent (64.71%) and N86/184F/D1246 was detected in 26.47% of the cases. The typical imported malaria case was middle-aged male, traveling from Angola, infected with P. falciparum carrying wild type pfmdr1 and pfk13. Our study highlights the need for constant surveillance of malaria parasites imported into Portugal as an important pillar of public health.

Anisakiasis is an emerging marine food-borne zoonosis resulting from the accidental ingestion of Anisakis larvae, through the consumption of raw or undercooked infected seafood products. The first case of human gastric hyperinfection by Anisakis simplex with an unusual and severe presentation, occurring in a Portuguese woman, is described in this article. Over 140 anisakid larvae were removed by gastroscopy. Massive infection is uncommon in areas where the consumption of raw fish is not part of the traditional diet, as is the case in Portugal. The increased consumption of raw seafood products is considered a health determinant in the rise in cases of anisakiasis. However, clinicians should be aware of the emergence of these infections, not only because of the new dietary habits of the population, but also because of the high prevalence of Anisakis larvae in the different fish species usually consumed by the population, collected on the Portuguese coast. Keywords: Anisakiasis, Foodborne disease, Fishery products, Emergent zoonosis

The Yellow Fever (YF) vaccination is recommended for people living in endemic areas and represents the most effective strategy to reduce the risk of infection. Previous studies have warned that booster regimens should be considered to guarantee the long-term persistence of 17DD-YF-specific memory components in adults living in areas with YF-virus circulation. Considering the lower seroconversion rates observed in children (9–12 months of age) as compared to adults, this study was designed in order to access the duration of immunity in single-dose vaccinated children in a 10-years cross-sectional time-span. The levels of neutralizing antibodies (PRNT) and the phenotypic/functional memory status of T and B-cells were measured at a baseline, 30–45 days, 1, 2, 4, 7, and 10 years following primary vaccination. The results revealed that a single dose induced 85% of seropositivity at 30–45 days and a progressive time-dependent decrease was observed as early as 2 years and declines toward critical values (below 60%) at time-spans of ≥4-years. Moreover, short-lived YF-specific cellular immunity, mediated by memory T and B-cells was also observed after 4-years. Predicted probability and resultant memory analysis emphasize that correlates of protection (PRNT; effector memory CD8+ T-cells; non-classical memory B-cells) wane to critical values within ≥4-years after primary vaccination. Together, these results clearly demonstrate the decline of 17DD-YF-specific memory response along time in children primarily vaccinated at 9–12 months of age and support the need of booster regimen to guarantee the long-term persistence of memory components for children living in areas with high risk of YF transmission.

Streptococcus canis is an animal pathogen which occasionally causes infections in humans. The S. canis M-like protein (SCM) encoded by the scm gene, is its best characterized virulence factor but previous studies suggested it could be absent in a substantial fraction of isolates. We studied the distribution and variability of the scm gene in 188 S. canis isolates recovered from companion animals (n = 152), wild animal species (n = 20), and humans (n = 14). Multilocus sequence typing, including the first characterization of wildlife isolates, showed that the same lineages are present in all animal hosts, raising the possibility of extensive circulation between species. Whole-genome analysis revealed that emm-like genes found previously in S. canis correspond to divergent scm genes, indicating that what was previously believed to correspond to two genes is in fact the same scm locus. We designed primers allowing for the first time the successful amplification of the scm gene in all isolates. Analysis of the scm sequences identified 12 distinct types, which could be divided into two clusters: group I (76%, n = 142) and group II (24%, n = 46) sharing little sequence similarity. The predicted group I SCM showed extensive similarity with each other outside of the N-terminal hypervariable region and a conserved IgG binding domain. This domain was absent from group II SCM variants found in isolates previously thought to lack the scm gene, which also showed greater amino acid variability. Further studies are necessary to elucidate the possible host interacting partners of the group II SCM variants and their role in virulence.

Identification of Aspergillus to species level is important since sibling species may display variable susceptibilities to multiple antifungal drugs and also because correct identification contributes to improve the knowledge of epidemiological studies. Two retrospective laboratory studies were conducted on Aspergillus surveillance at the Portuguese National Mycology Reference Laboratory. The first, covering the period 2017–2018, aimed to study the molecular epidemiology of 256 Aspergillus isolates obtained from patients with respiratory, subcutaneous, or systemic infections and from environmental samples. The second, using our entire collection of clinical and environmental A. fumigatus isolates (N = 337), collected between 2012 and 2019, aimed to determine the frequency of azole-resistant A. fumigatus isolates. Aspergillus fumigatus sensu stricto was the most frequent species in both clinical and environmental samples. Overall, and considering all Aspergillus sections identified, a high frequency of cryptic species was detected, based on beta-tubulin or calmodulin sequencing (37% in clinical and 51% in environmental isolates). Regarding all Fumigati isolates recovered from 2012–2019, the frequency of cryptic species was 5.3% (18/337), with the identification of A. felis (complex), A. lentulus, A. udagawae, A. hiratsukae, and A. oerlinghauensis. To determine the frequency of azole resistance of A. fumigatus, isolates were screened for azole resistance using azole-agars, and 53 possible resistant isolates were tested by the CLSI microdilution reference method. Nine A. fumigatus sensu stricto and six Fumigati cryptic isolates showed high minimal inhibitory concentrations to itraconazole, voriconazole, and/or posaconazole. Real-time PCR to detect cyp51A mutations and sequencing of cyp51A gene and its promoter were performed. The overall frequency of resistance to azoles in A. fumigatus sensu stricto was 3.0%. With this retrospective analysis, we were able to detect one azole-resistant G54R mutant A. fumigatus environmental isolate, collected in 2015. The TR34/L98H mutation, linked to environmental transmission route of azole resistance, was the most frequently detected mutation (N = 4; 1.4%). Our findings underline the demand for correct identification and susceptibility testing of Aspergillus isolates.

Sm16 is an immunomodulatory protein that seems to play a key role in the suppression of the cutaneous inflammatory response during Schistosoma mansoni penetration of the skin of definitive hosts. Therefore, Sm16 represents a potential target for protective immune responses induced by vaccination. In this work, we generated the recombinant protein rSm16 and produced polyclonal antibodies against this protein to evaluate its expression during different parasite life-cycle stages and its location on the surface of the parasite. In addition, we analyzed the immune responses elicited by immunization with rSm16 using two different vaccine formulations, as well as its ability to induce protection in Balb/c mice. In order to explore the biological function of Sm16 during the course of experimental infection, RNA interference was also employed. Our results demonstrated that Sm16 is expressed in cercaria and schistosomula and is located in the schistosomula surface. Despite humoral and cellular immune responses triggered by vaccination using rSm16 associated with either Freund’s or alum adjuvants, immunized mice presented no reduction in either parasite burden or parasite egg laying. Knockdown of Sm16 gene expression in schistosomula resulted in decreased parasite size in vitro but had no effect on parasite survival or egg production in vivo. Thus, our findings demonstrate that although the vaccine formulations used in this study succeeded in activating immune responses, these failed to promote parasite elimination. Finally, we have shown that Sm16 is not vital for parasite survival in the definitive host and hence may not represent a suitable target for vaccine development.

Background:Clostridium difficile infection (CDI) is prevalent in healthcare settings. The emergence of hypervirulent and antibiotic resistant strains has led to an increase in CDI incidence and frequent outbreaks. While the main virulence factors are the TcdA and TcdB toxins, antibiotic resistance is thought to play a key role in the infection by and dissemination of C. difficile.Methods: A CDI outbreak involving 12 patients was detected in a tertiary care hospital, in Lisbon, which extended from January to July, with a peak in February, in 2016. The C. difficile isolates, obtained from anaerobic culture of stool samples, were subjected to antimicrobial susceptibility testing with Etest®strips against 11 antibiotics, determination of toxin genes profile, PCR-ribotyping, multilocus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS).Results: Of the 12 CDI cases detected, 11 isolates from 11 patients were characterized. All isolates were tcdA-/tcdB+ and belonged to ribotype 017, and showed high level resistance to clindamycin, erythromycin, gentamicin, imipenem, moxifloxacin, rifampicin and tetracycline. The isolates belonged to four genetically related MLVA types, with six isolates forming a clonal cluster. Three outbreak isolates, each from a different MLVA type, were selected for WGS. Bioinformatics analysis showed the presence of several antibiotic resistance determinants, including the Thr82Ile substitution in gyrA, conferring moxifloxacin resistance, the substitutions His502Asn and Arg505Lys in rpoB for rifampicin resistance, the tetM gene, associated with tetracycline resistance, and two genes encoding putative aminoglycoside-modifying enzymes, aadE and aac(6′)-aph(2″). Furthermore, a not previously described 61.3 kb putative mobile element was identified, presenting a mosaic structure and containing the genes ermG, mefA/msrD and vat, associated with macrolide, lincosamide and streptogramins resistance. A substitution found in a class B penicillin-binding protein, Cys721Ser, is thought to contribute to imipenem resistance.Conclusion: We describe an epidemic, tcdA-/tcdB+, multidrug resistant clone of C. difficile from ribotype 017 associated with a hospital outbreak, providing further evidence that the lack of TcdA does not impair the infectious potential of these strains. We identified several determinants of antimicrobial resistance, including new ones located in mobile elements, highlighting the importance of horizontal gene transfer in the pathogenicity and epidemiological success of C. difficile.

The helminth Schistosoma mansoni is one of main causes of human schistosomiasis, a health and economic concern in some of the world's poorest countries. Current treatment regimens can lead to serious side effects and are not suitable for breastfeeding mothers. As such, efforts have been undertaken to develop a vaccine to prevent infection. Of these, Sm29 is a promising candidate that has been associated with resistance to infection/reinfection in humans and mice. Its ability to induce resistance to reinfection has also been recently demonstrated using a vaccine formulation containing Freund's adjuvant. However, Freund's adjuvant is unsuitable for use in human vaccines. We therefore evaluated the ability of Sm29 to induce protection against S. mansoni reinfection when formulated with either alum or MPLA as an adjuvant, both approved for human use. Our data demonstrate that, in contrast to Sm29 with MPLA, Sm29 with alum reduced parasite burden after reinfection compared to a control. We next investigated whether the immune response was involved in creating the differences between the protective (Sm29Alum) and non-protective (Sm29MPLA) vaccine formulations. We observed that both formulations induced a similar mixed-profile immune response, however, the Sm29 with alum formulation raised the levels of antibodies against Sm29. This suggests that there is an association between a reduction in worm burden and parasite-specific antibodies. In summary, our data show that Sm29 with an alum adjuvant can successfully protect against S. mansoni reinfection in mice, indicating a potentially effective vaccine formulation that could be applied in humans.

Although skin and soft tissue infections (SSTI) are the most common focal infections associated with invasive disease caused by Streptococcus pyogenes (Lancefield Group A streptococci - GAS), there is scarce information on the characteristics of isolates recovered from SSTI in temperate-climate regions. In this study, 320 GAS isolated from SSTI in Portugal were characterized by multiple typing methods and tested for antimicrobial susceptibility and SpeB activity. The covRS and ropB genes of isolates with no detectable SpeB activity were sequenced. The antimicrobial susceptibility profile was similar to that of previously characterized isolates from invasive infections (iGAS), presenting a decreasing trend in macrolide resistance. However, the clonal composition of SSTI between 2005 and 2009 was significantly different from that of contemporary iGAS. Overall, iGAS were associated with emm1 and emm3, while SSTI were associated with emm89, the dominant emm type among SSTI (19%). Within emm89, SSTI were only significantly associated with isolates lacking the hasABC locus, suggesting that the recently emerged emm89 clade 3 may have an increased potential to cause SSTI. Reflecting these associations between emm type and disease presentation, there were also differences in the distribution of emm clusters, sequence types, and superantigen gene profiles between SSTI and iGAS. According to the predicted ability of each emm cluster to interact with host proteins, iGAS were associated with the ability to bind fibrinogen and albumin, whereas SSTI isolates were associated with the ability to bind C4BP, IgA, and IgG. SpeB activity was absent in 79 isolates (25%), in line with the proportion previously observed among iGAS. Null covS and ropB alleles (predicted to eliminate protein function) were detected in 10 (3%) and 12 (4%) isolates, corresponding to an underrepresentation of mutations impairing CovRS function in SSTI relative to iGAS. Overall, these results indicate that the isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites, supporting a role for mutations impairing CovRS activity specifically in invasive infection and suggesting that this role relies on a differential regulation of other virulence factors besides SpeB.

Introdução: As infeções fúngicas superficiais são as dermatoses infeciosas mais frequentes e a sua incidência continua a aumentar. Os dermatófitos são os principais agentes causais apresentando, contudo, uma distribuição geográfica variável. Material e Métodos: O presente estudo teve como objetivo a caracterização epidemiológica das infeções fúngicas superficiais diagnosticadas nos Serviços/Unidades de Dermatologia pertencentes ao Serviço Nacional de Saúde Português entre janeiro de 2014 e dezembro 2016 através da análise retrospetiva dos resultados das culturas realizadas durante esse período. Resultados: Foram estudados 2375 isolamentos, pertencentes a 2319 doentes. O dermatófito mais frequentemente isolado foi o Trichophyton rubrum (53,6%), tendo sido o principal agente causal da tinha da pele glabra (52,4%) e das onicomicoses (51,1%). Relativamente às tinhas do couro cabeludo, globalmente o Microsporum audouinii foi o agente mais prevalente (42,6%), seguido do Trichophyton soudanense (22,1%). Enquanto na área metropolitana de Lisboa estes dermatófitos foram os principais agentes de tinha do couro cabeludo, nas regiões Norte e Centro o agente mais frequente foi o Microsporum canis (58,5%). Os fungos leveduriformes foram os principais responsáveis pelas onicomicoses das mãos (76,7%). Conclusão: Os resultados deste estudo estão globalmente concordantes com a literatura científica. O Trichophyton rubrum apresenta-se como o dermatófito mais frequentemente isolado em cultura. Na tinha do couro cabeludo, na área metropolitana de Lisboa, as espécies antropofílicas de importação assumem particular destaque.

In this investigation, machine-enhanced techniques were applied to bring about scientific insights to identify a minimum set of phenotypic/functional memory-related biomarkers for post-vaccination follow-up upon yellow fever (YF) vaccination. For this purpose, memory status of circulating T-cells (Naïve/early-effector/Central-Memory/Effector-Memory) and B-cells (Naïve/non-Classical-Memory/Classical-Memory) along with the cytokine profile (IFN/TNF/IL-5/IL-10) were monitored before-NV(day0) and at distinct time-points after 17DD-YF primary vaccination-PV(day30-45); PV(year1-9) and PV(year10-11). A set of biomarkers (eEfCD4; EMCD4; CMCD19; EMCD8; IFNCD4; IL-5CD8; TNFCD4; IFNCD8; TNFCD8; IL-5CD19; IL-5CD4) were observed in PV(day30-45), but not in NV(day0), with most of them still observed in PV(year1-9). Deficiencies of phenotypic/functional biomarkers were observed in NV(day0), while total lack of memory-related attributes was observed in PV(year10-11), regardless of the age at primary vaccination. Venn-diagram analysis pre-selected 10 attributes (eEfCD4, EMCD4, CMCD19, EMCD8, IFNCD4, IL-5CD8, TNFCD4, IFNCD8, TNFCD8 and IL-5CD4), of which the overall mean presented moderate accuracy to discriminate PV(day30-45)&PV(year1-9) from NV(day0)&PV(year10-11). Multi-parameter approaches and decision-tree algorithms defined the EMCD8 and IL-5CD4 attributes as the top-two predictors with moderated performance. Together with the PRNT titers, the top-two biomarkers led to a resultant memory status observed in 80% and 51% of volunteers in PV(day30-45) and PV(year1-9), contrasting with 0% and 29% found in NV(day0) and PV(year10-11), respectively. The deficiency of memory-related attributes observed at PV(year10-11) underscores the conspicuous time-dependent decrease of resultant memory following17DD-YF primary vaccination that could be useful to monitor potential correlates of protection in areas under risk of YF transmission.

Introduction The diagnosis of schistosomiasis mansoni on early stages of infection is important to prevent late morbidity. A simple, cheap, sensitive and specific assay for routine diagnosis of schistosome infection based on the detection of specific IgG for schistosomula tegument antigens (ELISA-SmTeg) was developed by our group. Methods We describe here an acute outbreak involving a travel group of 80 individuals from a non-endemic area of the State of Minas Gerais, Brazil. These individuals were in contact with a freshwater pool where Biomphalaria glabrata was found. Results obtained from our new methodology were compared to IgG antibody titers against soluble worm antigenic preparation (SWAP) by ELISA and, also to parasitological examination, nuclear magnetic resonance and clinical findings. Results ELISA-SmTeg was capable of detecting 64 positive cases among the 80 individuals participating at the survey with a positivity ratio of 80% and a higher sensitivity than ELISA-SWAP that was only sensitive for 56% of positive cases. Besides, a significant correlation was found for the severity of the infection and the specific IgG titers against SmTeg. Conclusions Our data showed that ELISA-SmTeg might serve as the initial diagnostic tool for acute stages of the infection in community-based helminth control programs or for the surveillance of individuals from non-endemic areas.

The development of a more sensitive diagnostic test for schistosomiasis is needed to overcome the limitations of the use of stool examination in low endemic areas. Using parasite antigens in enzyme linked immunosorbent assay is a promising strategy, however a more rational selection of parasite antigens is necessary. In this study we performed in silico analysis of the Schistosoma mansoni genome, using SchistoDB database and bioinformatic tools for screening immunogenic antigens. Based on evidence of expression in all parasite life stage within the definitive host, extracellular or plasmatic membrane localization, low similarity to human and other helminthic proteins and presence of predicted B cell epitopes, six candidates were selected: a glycosylphosphatidylinositol-anchored 200 kDa protein, two putative cytochrome oxidase subunits, two expressed proteins and one hypothetical protein. The recognition in unidimensional and bidimensional Western blot of protein with similar molecular weight and isoelectric point to the selected antigens by sera from S. mansoni infected mice indicate a good correlation between these two approaches in selecting immunogenic proteins.

Mycoplasma sinoviae is a major pathogen of poultry causing synovitis and respiratory infection. M. synoviae hemagglutinin (VlhA) is a lipoprotein encoded by related multigene families that appear to have arisen by horizontal gene transfer. It is an abundant immunodominant surface protein involved in host-parasite interaction mediating binding to host erythrocytes. Herein, we have performed in silico analysis of the vlhA gene product from the Mycoplasma synoviae 53 strain and compared it to the VlhA protein of M. synoviae WUV1853 strain. The VlhA of the M. synoviae 53 strain possesses 569 amino acids and showed 85% identity with the VlhA protein of the M. synoviae WUV1853 strain. Further, a signal peptide was identified from amino acid M1 to D28 and a cleavage site between D28 and Q29, both located in the N-terminal domain of the molecule. Additionally, an insertion of PAPT amino acids was observed between T30-P35 and a deletion of the amino acids GTPGNP within the PRR region of the VlhA from the M. synoviae 53 strain, which may be related to its reduced virulence. Finally, we have identified 17 B cell epitopes and 22 T cells epitopes within the VlhA from the M. synoviae 53 strain. The B cell epitope S263-D277 and the T cell epitopes N45-N54 and G58-N67 showed 100% and 87-100% identity, respectively, with regions of VlhA protein of tested Mycoplasma synoviae and Mycoplasma galisepticum strains. Thus, these peptides represent new candidate molecules for the development of efficient diagnostic assays and new subunit vaccines.

The successful development of vaccines depend on the knowledge of the immunological mechanisms associated with the elimination of the pathogen. In the case of schistosomes, its complex life cycle and the mechanisms developed to evade host immune system, turns the development of a vaccine against the disease into a very difficult task. Identifying the immunological effector mechanisms involved in parasite attrition and the major targets for its response is a key step to formulate an effective vaccine. Recent studies have described some promising antigens to compose a subunit vaccine and have pointed to some immune factors that play a role in parasite elimination. Here we review the immune components and effector mechanisms associated with the protective immunity induced by those vaccine candidates and the lessons we have learned from the studies of the acquired resistance to infection in humans. We will also discuss the immune factors that correlate with protection and therefore could help to evaluate those vaccine formulations in clinical trials.

BACKGROUND:A vaccine against schistosomiasis would have a great impact in disease elimination. Sm29 and Sm22.6 are two parasite tegument proteins which represent promising antigens to compose a vaccine. These antigens have been associated with resistance to infection and reinfection in individuals living in endemic area for the disease and induced partial protection when evaluated in immunization trials using naïve mice. METHODOLOGY/PRINCIPALS FINDINGS:In this study we evaluated rSm29 and rSm22.6 ability to induce protection in Balb/c mice that had been previously infected with S. mansoni and further treated with Praziquantel. Our results demonstrate that three doses of the vaccine containing rSm29 were necessary to elicit significant protection (26%-48%). Immunization of mice with rSm29 induced a significant production of IL-2, IFN-γ, IL-17, IL-4; significant production of specific antibodies; increased percentage of CD4+ central memory cells in comparison with infected and treated saline group and increased percentage of CD4+ effector memory cells in comparison with naïve Balb/c mice immunized with rSm29. On the other hand, although immunization with Sm22.6 induced a robust immune response, it failed to induce protection. CONCLUSION/SIGNIFICANCE:Our results demonstrate that rSm29 retains its ability to induce protection in previously infected animals, reinforcing its potential as a vaccine candidate.

Schistosomiasis is a debilitating disease that represents a major health problem in at least 74 tropical and subtropical countries. Current disease control strategies consist mainly of chemotherapy which cannot prevent recurrent re-infection of people living in endemic area. In the last decades, many researchers made a remarkable effort in the search for an effective vaccine to provide long-term protection. Parasitic platyhelminths of Schistosoma genus, which cause the disease, live in the blood vessels of definitive hosts where they are bathed in host blood for many years. Among the most promising molecules as vaccine candidates are the proteins present in the host-parasite interface, so numerous tegument antigens have been assessed and the achieved protection never got even close to 100%. Besides the tegument, the digestive tract is the other major site of host-parasite interface. Since parasites feed on blood, they need to swallow a considerable amount of blood for nutrient acquisition. Host blood ingested by schistosomes passes through the esophagus and reaches the gut where many peptidases catalyze the proteolysis of blood cells. Recent studies show the emergence of antigens related to the parasite blood feeding, such as esophageal gland proteins, proteases and other proteins related to nutrient uptake. Herein, we review what is known about S. mansoni digestive tract proteins, emphasizing the ones described as potential vaccine candidates.

Introdução: A infeção do trato urinário é a complicação infeciosa mais comum no período pós transplante renal, estando a sua frequência pouco caracterizada na população portuguesa. Este trabalho teve como objetivo determinar a incidência de infeções do trato urinário e infeções do trato urinário recorrentes em transplantados renais. Material e Métodos: Tratou-se de um estudo observacional de coorte retrospetiva, com consulta dos processos clínicos de doentes transplantados entre Janeiro de 2004 e Dezembro de 2005, no Hospital de Santa Cruz, com seguimento durante cinco anos ou até à data de perda de enxerto, morte ou perda de follow-up. Após uma análise descritiva da população, utilizámos testes bivariados para identificação de fatores associados a infeções do trato urinário. Resultados: Em 127 doentes incluídos com seguimento de 593 doentes/ ano, 53 (41,7%) tiveram pelo menos um episódio de infeção do trato urinário e 21 (16,5%) tiveram infeções do trato urinário recorrentes. O género feminino foi o único fator associado com ocorrência de infeções do trato urinário (p < 0,001, OR = 7,08, RR = 2,95) e infeções do trato urinário recorrentes (p < 0,001, OR = 4,66, RR = 2,83). Os agentes etiológicos mais frequentes foram Escherichia coli (51,6%), Klebsiella pneumoniae (15,5%) e Enterobacter spp (9,9%). As infeções do trato urinário não causaram aumento de mortalidade ou perda de enxerto, mas foram a principal causa de internamentos hospitalares. Discussão: Na nossa população, apenas o género feminino foi identificado como fator de risco para o desenvolvimento de infeções do trato urinário, recorrentes ou não. Escherichia coli foi o agente etiológico mais frequente. Conclusão: Apesar das medidas preventivas adotadas, as infeções do trato urinário continuam a ser uma importante causa de morbilidade e de internamentos hospitalares. Palavras-chave: Infeção do Tracto Urinário; Complicações Pós-operatórias; Factores de Risco; Transplantação Renal; Portugal.

The development of a vaccine against schistosomiasis and also the availability of a more sensitive diagnosis test are important tools to help chemotherapy in controlling disease transmission. Bioinformatics tools, together with the access to parasite genome, published recently, should help generate new knowledge on parasite biology and search for new vaccines or therapeutic targets and antigens to be used in the disease diagnosis. Parasite surface proteins, especially those expressed in schistosomula tegument, represent interesting targets to be used in vaccine formulations and in the diagnosis of early infections, since the tegument represents the interface between host and parasite and its molecules are responsible for essential functions to parasite survival. In this paper we will present the advances in the development of vaccines and diagnosis tests achieved with the use of the information from schistosome genome focused on parasite tegument as a source for antigens.