Long-term monitoring of COVID-19 prevalence in raw and treated wastewater in Salvador, the largest capital of the Brazilian Northeast.

Wastewater-based epidemiology (WBE) becomes an interesting epidemiological approach to monitoring the prevalence of SARS-CoV-2 broadly and non-invasively. Herein, we employ for the first time WBE, associated or not with the PEG 8000 precipitation method, for the detection of SARS-CoV-2 in samples of raw or treated wastewater from 22 municipal wastewater treatment stations (WWTPs) located in Salvador, the fourth most populous city in Brazil. Our results demonstrate the success of the application of WBE for detecting SARS-CoV-2 in both types of evaluated samples, regardless of the usage of PEG 8000 concentration procedure. Further, an increase in SARS-CoV-2 positivity rate was observed in samples collected in months that presented the highest number of confirmed COVID-19 cases (May/2021, June/2021 and January/2022). While PEG 8000 concentration step was found to significantly increase the positivity rate in treated wastewater samples (p < 0.005), a strong positive correlation (r: 0.84; p < 0.002) between non-concentrated raw wastewater samples with the number of new cases of COVID-19 (April/2021-February/2022) was observed. In general, the present results reinforce the efficiency of WBE approach to monitoring the presence of SARS-CoV-2 in either low- or high-capacity WWTPs. The successful usage of WBE even in raw wastewater samples makes it an interesting low-cost tool for epidemiological surveillance.

Detection of Hemopathogens in Chelonoidis carbonaria: Microscopic, Molecular, Hematological, and Clinical Biochemistry Aspects.

Background: The growing contact between men and wild animals, caused by the increase in the population in urban centers and the destruction of the habitat of these animals, has been leading to a greater circulation of pathogens between humans and wildlife. Chelonoidis carbonaria, a tortoise found throughout South America, is one of the animals most rescued from animal trafficking and illegal breeding. Considering this situation, this study aimed to verify the occurrence of hemoparasites in C. carbonaria. Materials and Methods: Blood samples from 73 C. carbonaria were collected from animals located in (1) a rural commercial breeding unit, (2) an urban zoo, and (3) a center of rescued animal screening. Genomic DNA was extracted from these animals and used in PCRs to detect specific genomic fragments of haemogregarines (i.e., Hepatozoon and Hemolivia), and members of the Anaplasmataceae Family (i.e., Ehrlichia sp. and Anaplasma sp.). Blood samples were screened for hemopathogens by direct microscopy and were used for hematological assays, and serum samples were analyzed to determine the concentration of serum components. Results: It was found that 34.2% of the tortoises presented Sauroplasma sp. in their blood samples; these animals showed clinical biochemistry changes that indicate altered liver function. Two zoo animals were positive for Ehrlichia sp. in PCR, and also presented clinical biochemistry and hematological changes. Conclusion: The present project is pioneer in the detection of Ehrlichia sp. in C. carbonaria, and was able to identify changes in clinical biochemistry that can be a result of the infection by hemopathogens in this species.

First description of Candida haemulonii infecting a snake Boa constrictor: Molecular, pathological and antifungal sensitivity characteristics.

Candida haemulonii is an emergent infectious pathogen that affects human presenting comorbidities and/or immunodepression. Little is known about other possible hosts. For the first time, this fungus was found causing a cutaneous infection in a snake, Boa constrictor, characterized by scale opacity and several ulcerative lesions. This C. haemulonii was isolated, identified using molecular techniques and a phylogenetic study, and had its growth totally inhibited by all the drugs tested; however, no fungicide effect was seen for fluconazole and itraconazole. The B. constrictor clinical signals subsided after a treatment using a biogenic silver nanoparticle-based ointment. These findings, along with the B. constrictor presence near human habitats, warn for the necessity of wildlife health monitoring for emergent and opportunistic diseases in peri-urban environments.

Activity of Fusarium oxysporum-Based Silver Nanoparticles on Candida spp. Oral Isolates.

Candida spp. resistant to commercially available antifungals are often isolated from patients with oral candidiasis, a situation that points to the need for the development of new therapies. Thus, we evaluated the activity of Fusarium oxysporum-based silver nanoparticles (AgNPs) on Candida spp. isolated from denture stomatitis lesions. Candida isolates were molecularly identified and submitted to susceptibility assays using AgNPs and commercial fungicides. The interference on biofilm formation and the mechanisms of action of AgNPs on Candida spp. were also investigated. Scanning electron microscopy was used to evaluate the morphology of AgNP-treated Candida. Candida albicans was the most frequent species isolated from denture stomatitis cases. All Candida spp. were susceptible to AgNPs at low concentrations, except Candida parapsilosis. AgNPs caused surface damage, cell disruption, and biofilm formation inhibition. The ergosterol supplementation protected C. albicans against the AgNP action. AgNPs are effective against Candida spp. and can be faced as a promising new therapeutic agent against oral candidiasis.

Evaluation of SARS-CoV-2 concentrations in wastewater and river water samples.

There are only a few established methods to determine the concentration of encapsulated viruses, such as SARS-CoV-2, in water matrices, limiting the application of wastewater-based epidemiology (WBE)-an important tool for public health research. The present study compared four methods that are commonly used to concentrate non-encapsulated enteric viruses for determining SARS-CoV-2 concentration in wastewater and wastewater-enriched river water samples. The four methods tested were electronegative membrane with Mg+2 addition, aluminum hydroxide-based precipitation, polyethylene glycol (PEG) 8000 precipitation, and ultrafiltration (with porosity of 10 and 50 kDa). Prior to the concentration step, filtration or centrifugation was performed to remove suspended particles from the samples (pretreatment). To evaluate the recovery efficiency (%), samples of SARS-CoV-2 from nasopharyngeal swabs obtained from RT-qPCR-positive patients were used as spiked samples. The second part of the analysis involved the quantification of the SARS-CoV-2 copy number in analytes without SARS-CoV-2-spiked samples. Among the tested methods, pretreatment via centrifugation followed by ultrafiltration with a 50-kDa cut-off was found the most efficient method for wastewater samples with spiked samples (54.3 or 113.01% efficiency). For the wastewater-enriched river samples with spiked samples, pretreatment via centrifugation followed by filtration using an electronegative membrane was the most efficient method (110.8% and 95.9% for N1 and N2 markers, respectively). However, ultrafiltration of the raw river water samples using 10 or 50 kDa cut-off filters and PEG 8000 precipitation showed the best concentration efficiency based on copy number, regardless of the pretreatment approach or sample type (values ranging from 3 × 105 to 6.7 × 103). The effectiveness of the concentration method can vary depending on the type of sample and concentration method. We consider that this study will contribute to more widespread use of WBE for the environmental surveillance of SARS-CoV-2.

Activity of antifungal drugs and Brazilian red and green propolis extracted with different methodologies against oral isolates of Candida spp.

BACKGROUND: Oral candidiasis is an opportunistic disease caused by fungi of the Candida genus. The occurrence of Candida spp. resistance to the commercial antifungal drugs points to the search for alternative treatments. Propolis has been successfully used in the treatment of infectious diseases for centuries. It has been proposed that an ultrasound pretreatment in the propolis extraction protocol can enhance the concentrations of molecules with antimicrobial activities in the final extract. Thus, this study aimed to compare the antifungal activity against oral Candida spp. isolates of green and red propolis extracts submitted or not to an ultrasound pretreatment before the extraction procedure. METHODS: Candida spp. were isolated from denture stomatitis lesions and identified by sequencing. Oral Candida spp. isolates and reference strains were submitted to broth microdilution assays using commercial antifungals and Brazilian green and red propolis extracts submitted or not to an ultrasound pretreatment. Minimal Inhibitory Concentrations (MIC) and Minimal Fungicide Concentrations (MFC) were determined and biofilm formation interference was evaluated for resistant isolates. RESULTS: C. albicans, Candida tropicalis and Candida dubliniensis were isolated from denture stomatitis lesions. Growth inhibition was observed in all Candida isolates incubated with all green and red propolis extracts. At lower doses, red propolis extracts presented significant antifungal activity. The ultrasound pretreatment did not promote an increase in the antifungal activity of green or red propolis. Three isolates, which were highly resistant to fluconazole and itraconazole, were susceptible to low doses of red propolis extracts. These same three specimens had their biofilm formation inhibted by red propolis ethanolic extract. CONCLUSIONS: Thus, red propolis can be faced as a promising natural product to be used in the auxiliary antifungal therapy of denture stomatitis.

Applications of Silver Nanoparticles in Dentistry: Advances and Technological Innovation.

Silver nanoparticles (AgNPs) have been successfully applied in several areas due to their significant antimicrobial activity against several microorganisms. In dentistry, AgNP can be applied in disinfection, prophylaxis, and prevention of infections in the oral cavity. In this work, the use of silver nanoparticles in dentistry and associated technological innovations was analyzed. The scientific literature was searched using PubMed and Scopus databases with descriptors related to the use of silver nanoparticles in dentistry, resulting in 90 open-access articles. The search for patents was restricted to the A61K code (International Patent Classification), using the same descriptors, resulting in 206 patents. The results found were ordered by dental specialties and demonstrated the incorporation of AgNPs in different areas of dentistry. In this context, the search for patents reaffirmed the growth of this technology and the dominance of the USA pharmaceutical industry over AgNPs product development. It could be concluded that nanotechnology is a promising area in dentistry with several applications.

Leishmania infantum-Derived Glycoinositolphospholipids in the Immunodiagnosis of Subclinically Infected Dogs.

Lipophosphoglycan (LPG), when used as an ELISA target, confers high specificity and sensitivity to the detection of Leishmania infantum antibodies in dogs. Glycoconjugates are economically viable because the yield is very high after extraction/purification. In addition, they are very stable, which allows their use in point-of-care testing without special storage conditions. During the glycoconjugate extraction, a glycoinositolphospholipid (GIPL)-enriched fraction is obtained in similar quantities as LPG. Since GIPLs can be extracted from the same parasite pellet as LPGs, this work aimed to evaluate the immune recognition of GIPLs by Leishmania infantum-infected dogs and its use for canine leishmaniasis (CanL) immunodiagnosis. Like LPG, GIPLs were recognized by sera from L. infantum-infected dogs, but with less sensitivity (83.8%). However, 80% (16/20) of subclinically infected dogs were detected as positive in the assay. Different from LPG, the GIPL-based assay achieved a lower specificity (73.7%) and cross-reactions occurred with T. cruzi and L. braziliensis-infected dogs. Although GIPLs exhibited a similar performance to LPG for subclinically L. infantum-infected dogs, the occurrence of cross-reactivities with other protozoa and a lower sensitivity hinders its use for an immunodiagnostic test. In places where those diseases do not co-exist such as in the Mediterranean region, its use for subclinically dogs could be an alternative.

Leptospira interrogans in wild Boa constrictor snakes from Northeast Brazil peri­urban rainforest fragments.

Leptospirosis, a disease that occurs worldwide, especially in tropical regions, is caused by bacteria of the genus Leptospira and affects mammals, amphibians, and reptiles. Boa constrictor snakes are commonly found in Atlantic rainforest fragments in peri­urban areas, which indicates a greater possibility of the contact of these animals with humans residing there. Therefore, the aim of this work was to detect Leptospira spp infection through molecular assays in wild B. constrictor snakes rescued in peri­urban areas and verify seroreactivity, by the microscopic agglutination test (MAT), as well as the most common serogroups. Among the 46 samples tested, 7 (15.21%) were positive according to PCR and confirmed as Leptospira interrogans through secY gene sequencing. In MAT, 37 (80.43%) of the 46 samples were classified as reactive. Panama was the serogroup with the highest occurrence. The results showed the presence of Leptospira spp DNA in asymptomatic snakes rescued in rainforest fragments located in peri­urban areas and support further investigations on the influence of these animals in the epidemiology of leptospirosis in tropical peri­urban areas.

Ticks and Associated Pathogens From Rescued Wild Animals in Rainforest Fragments of Northeastern Brazil.

The Ixodidae family comprises ticks that are hematophagous ectoparasites and are considered vectors of several hemoparasites from the Anaplasmataceae family and the genus Hepatozoon, Babesia, and Rickettsia. These ectoparasites parasitize domestic and wild animals belonging to several vertebrate groups. Ticks are highly adapted to different biomes and thus possess a wide geographical distribution. In the Brazilian state of Bahia, localized in the Northeast region, there are large rainforest fragments. Studies have rarely been carried out on ticks, and their hemoparasites, that parasitize wild animals in this region. Thus, this study aimed to identify the tick species parasitizing wild animals rescued in rainforest fragments of Bahia and investigate the presence of hemoparasites in tick tissues. During a 2-year period, 238 ticks were collected from 41 wild mammalians, reptiles, and amphibians. These ectoparasites were taxonomically classified according to their morphological characteristics. The ticks identified belonged to five different species from the Ixodidae family: Amblyomma varium, Amblyomma rotundatum, Amblyomma nodosum, Ixodes loricatus, and Rhipicephalus sanguineus. For the first time, an A. rotundatum parasitizing the Mesoclemmys tuberculata turtle was described. PCR assays using DNA extracted from salivary glands or midgut of the ticks were performed to detect specific DNA fragments of hemoparasites from the genus Rickettsia, Ehrlichia, Babesia, Hepatozoon, and from the Anaplasmataceae family. The results showed positive detection of the Rickettsia genus (7.9%), Anaplasmataceae family (15.8%), and Hepatozoon genus (15.8%). Specific DNA from the Ehrlichia and Babesia genera were not detected in these samples. Specific DNA from members of the Anaplasmataceae family was detected in A. varium for the first time. The present work showed that amphibians, reptiles, and mammals from Bahia's Atlantic Forest areparasitized by different tick species, and that these ectoparasites present pathogens in their tissues that impact both humans and animals due to their zoonotic potential.

Susceptibility of Malassezia pachydermatis Clinical Isolates to Allopathic Antifungals and Brazilian Red, Green, and Brown Propolis Extracts.

Clinical mycoses treatment is associated with issues such as negative side effects, high cost, prolonged treatment, and resistant strain selection. Malassezia pachydermatis is the most frequently isolated yeast in cases of canine otitis and dermatitis. The number of fungal strains exhibiting primary resistance to several drugs in vitro is increasing. Propolis has a diverse chemical composition and well-known therapeutic properties against mycoses. An alternative method for producing propolis extracts using supercritical fluid has higher selectivity, yielding extracts with fewer pollutant residues. This study therefore aimed to evaluate the in vitro susceptibility profile of M. pachydermatis clinical isolates to precharacterized supercritical and ethanolic extracts. Three types of Brazilian propolis extracts (green, red, and brown) and commercial allopathic antifungals were used in this investigation. We used the microdilution broth technique to evaluate the susceptibility profile of the yeasts. The minimum inhibitory concentration (MIC) of the brown propolis ethanolic extract was ≥16 µg/mL for all isolates. The MICs of fluconazole, ketoconazole, itraconazole, and amphotericin B ranged from 8 to >64 µg/mL, 0.032-4 µg/mL, 0.0313-16 µg/mL, and 1-2 µg/mL, respectively. The MICs of ethanolic red propolis extracts were lower than those of supercritical red propolis extracts. However, the green propolis ethanolic extract had more pronounced fungicidal activity. Isolates with lower susceptibility to commercial fungicides were inhibited by red and green propolis extracts. These results indicate that propolis can potentially be used in in vivo experiments as a promising therapeutic agent against M. pachydermatis infections.