Acanthamoeba keratitis (AK) is a painful and potentially blinding corneal infection caused by Acanthamoeba spp. In Madrid, environmental studies have demonstrated a high presence of these free-living amoebae in tap water. Since most of AK cases occur in contact lenses (CL) wearers with inadequate hygiene habits, the presence of Acanthamoeba in discarded CL has been studied and compared with other common etiological agents of keratitis, such as Pseudomonas aeruginosa and Staphylococcus aureus. One hundred and seventy-seven healthy individuals from Madrid contributed their discarded CL and answered a questionnaire on hygiene habits. DNA was extracted from the CL solution and analyzed by real-time PCR for Acanthamoeba, Pseudomonas aeruginosa and Staphylococcus aureus. These CL and their solutions were also cultured on non-nutrient agar to isolate Acanthamoeba. Among the 177 samples, Acanthamoeba DNA was detected in 87 (49.2%), P. aeruginosa DNA in 14 (7.9%) and S. aureus DNA in 19 (10.7%). Cultivable amoebae, however, were observed in only one sample (0.6%). This isolate was genotyped as T4. The habits reported by this CL owner included some recognized risk factors for AK, but in this study only the practice of “not cleaning the CL case” presented some statistical significant association with Acanthamoeba DNA presence. Detection of the investigated bacterial DNA did not demonstrate statistical significant association with the studied practices, but the presence of P. aeruginosa revealed a possible inhibition of Acanthamoeba in these samples. The PCR results suggest a high presence of Acanthamoeba spp. in healthy CL wearers from Madrid, but we can assume that CL solutions are properly disinfecting the CL since only 1.1% of the positive PCR samples correspond to viable amoebae and, after four years, only one participant reported stronger ocular problems. Nevertheless, more studies are necessary to corroborate this hypothesis.

Legionnaires’ disease is a severe form of pneumonia, with worldwide relevance, caused by Legionella spp. Approximately 90% of all cases of legionellosis are caused by Legionella pneumophila, but other species can also be responsible for this infection. These bacteria are transmitted by inhalation of aerosols or aspiration of contaminated water. In Spain, environmental studies have demonstrated the presence of Legionella non-pneumophila species in drinking water treatment plants and water distribution networks. Aware that this evidence indicates a risk factor and the lack of routine assays designed to detect simultaneously diverse Legionella species, we analyzed 210 urine samples from patients presenting clinical manifestations of pneumonia using a semi-nested PCR for partial amplification of the 16S rDNA gene of Legionella and a diagnostic method used in hospitals for Legionella antigen detection. In this study, we detected a total of 15 cases of legionellosis (7.1%) and the first case of Legionnaires’ disease caused by L. anisa in Spain. While the conventional method used in hospitals could only detect four cases (1.9%) produced by L. pneumophila serogroup 1, using PCR, the following species were identified: Legionella spp. (10/15), L. pneumophila (4/15) and L. anisa (1/15). These results suggest the need to change hospital diagnostic strategies regarding the identification of Legionella species associated with this disease. Therefore, the detection of Legionella DNA by PCR in urine samples seems to be a suitable alternative method for a sensitive, accurate and rapid diagnosis of Legionella pneumonia, caused by L. pneumophila and also for L. non-pneumophila species.

The genus Legionella comprises more than 60 species, and about half are associated with infection. Legionella pneumophila is the most commonly associated with these infections and by far the most studied, but L. non-pneumophila species, such as L. feeleii, L. anisa, etc., may also present clinical importance. Free-living amoebae are their preferred environmental host, where these bacteria not only survive but also succeed in multiplying, and this relationship can lead to an increase in bacterial virulence. The goal of this study was to evaluate the alterations of Legionella pathogenicity due to its interaction with Acanthamoeba. For this, the expression of protein effectors SdhA, LegK2, and SidK were evaluated in L. pneumophila and L. feeleii, before and after infecting Acanthamoeba. Additionally, the host response was evaluated by measuring the production of IL-6, IL-8, and IFN-γ in infected macrophages. Regarding the virulence factors, an increase in SdhA expression was observed after these bacteria infected Acanthamoeba, with a higher increase in the macrophage cultures infected with L. feeleii. Also, an increase in the expression of LegK2 was observed after infecting Acanthamoeba, but it was more intense in the cultures infected with L. pneumophila. With regard to SidK, it was increased in L. feeleii after infecting Acanthamoeba, however the same effect was not observed for L. pneumophila. In cytokine production, the effect on IL-6 and IL-8 was similar for both cytokines, increasing their concentration, but higher production was observed in the cultures infected with L. feeleii, even though it demonstrated slightly lower production with the inoculum obtained from Acanthamoeba. Concerning IFN-γ, induction was observed in both species but higher in the infection by L. pneumophila. Nevertheless, it is not known if this induction is enough to promote an efficient immune response against either L. pneumophila or L. feeleii. Altogether, these alterations seem to increase L. feeleii virulence after infecting Acanthamoeba. However, this increase does not seem to turn L. feeleii as virulent as L. pneumophila. More studies are necessary to understand the aspects influenced in these bacteria by their interaction with Acanthamoeba and, thus, identify targets to be used in future therapeutic approaches.

Lynx pardinus is one of the world’s most endangered felines inhabiting the Iberian Peninsula. The present study was performed to identify the presence of microsporidia due to the mortality increase in lynxes. Samples of urine (n = 124), feces (n = 52), and tissues [spleen (n = 13), brain (n = 9), liver (n = 11), and kidney (n = 10)] from 140 lynxes were studied. The determination of microsporidia was evaluated using Weber’s chromotrope stain and Real Time-PCR. Of the lynxes analyzed, stains showed 10.48% and 50% positivity in urine and feces samples, respectively. PCR confirmed that 7.69% and 65.38% belonged to microsporidia species. The imprints of the tissues showed positive results in the spleen (38.46%), brain (22.22%), and liver (27.27%), but negative results in the kidneys. PCR confirmed positive microsporidia results in 61.53%, 55.55%, 45.45%, and 50%, respectively. Seroprevalence against Encephalitozoon cuniculi was also studied in 138 serum samples with a positivity of 55.8%. For the first time, the results presented different species of microsporidia in the urine, feces, and tissue samples of Lynx pardinus. The high titers of anti-E. cuniculi antibodies in lynx sera confirmed the presence of microsporidia in the lynx environment. New studies are needed to establish the impact of microsporidia infection on the survival of the Iberian lynx.

Aptamer development has traditionally relied on iterative, labor-intensive experimental approaches. Recent advances in bioinformatics and computational power now enable the rational in silico design of high-affinity nucleic acid ligands, drastically reducing time and cost. Here, we present an integrated computational pipeline for designing ssDNA aptamers targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike (S) protein. A library of 4000 randomly generated 20-nucleotide sequences with defined base ratios (C:G:A:T = 3:3:2:2) was screened based on secondary structure and free energy. The 100 most stable sequences (ΔG ≤ −4.1 kcal/mol) were modeled in three dimensions, yielding 1000 conformations. The 100 structures with the lowest potential energies (≤−78,051.7 kcal/mol) were selected for molecular docking with the RBD in the closed, open, and transitional states. Two aptamers—model-2-seq_2714 (seq_2714) and model-5-seq_1397 (seq_1397)—were selected for experimental assessment based on their favorable predicted docking scores against the RBD. Western blot and dot blot assays showed detectable recognition of the recombinant RBD by both aptamers under the experimental conditions tested. Exploratory testing in a limited number of clinical and environmental samples further showed that detectable signals could be obtained in selected complex matrices. These findings provide an initial proof-of-principle assessment of a computational-first strategy for prioritizing candidate aptamers for subsequent experimental testing.