SARS-CoV-2, the novel Coronavirus, was first detected in Wuhan, China, in December 2019, and has since spread rapidly, causing millions of deaths worldwide. As in most countries of the world, in Brazil, the consequences of the COVID-19 pandemic have been catastrophic. Several studies have reported the fecal shedding of SARS-CoV-2 RNA titers from infected symptomatic and asymptomatic individuals. Therefore, the quantification of SARS-CoV-2 in wastewater can be used to track the virus spread in a population. In this study, samples of untreated wastewater were collected for 44 weeks at five sampling sites in the ABC Region (São Paulo, Brazil), in order to evaluate the SARS-CoV-2 occurrence in the sewerage system. SARS-CoV-2 RNA titers were detected throughout the period, and the concentration ranged from 2.7 to 7.7 log 10 genome copies.L −1 , with peaks in the last weeks of monitoring. Furthermore, we observed a positive correlation between the viral load in wastewater and the epidemiological/clinical data, with the former preceding the latter by approximately two weeks. The COVID-19 prevalence for each sampling site was estimated via Monte-Carlo simulation using the wastewater viral load. The mean predicted prevalence ranged 0.05 to 0.38%, slightly higher than reported (0.016 ± 0.005%) in the ABC Region for the same period. These results highlight the viability of the wastewater surveillance for COVID-19 infection monitoring in the largest urban agglomeration in South America. This approach can be especially useful for health agencies and public decision-makers in predicting SARS-CoV-2 outbreaks, as well as in local tracing of infection clusters.

There is a growing necessity to integrate morphological and genetic studies. This paper proposes a new technique that allows DNA extraction of arthropods while still keeping intact the entire morphology of the specimens. The technique uses Proteinase K to dissolve protein tissues and preserve the chitinous exoskeleton of specimens. The method is fast, cheap, non-toxic, and allows for good morphological preparations of specimens retaining much of their tridimensional structure. The methodology works fine with specimens preserved in different kinds of media, such as for dry (pinned) specimens, and specimens preserved in Ethanol. In addition, it allows the extraction of DNA from fresh specimens, as well as from specimens preserved for a long time. The technique works well for morphological studies alone, but allows the generation of an associated genomic library at an individual-scale. Among the advantages of the new technique is the possibility of extracting DNA from the entire specimen (necessary for the study of diseases transmitted by arthropod vectors), while still keeping the morphology intact for correct taxonomic identification. In addition, in comparison with methods that extract DNA from small tissue samples (e.g., from legs or wings), the method allows for the extraction of a larger amount of DNA and is better suited for small specimens.

There are currently no reports on the isolation and molecular examination of Toxoplasma gondii from bats. Here, we report the isolation and genotypic characterisation of two T. gondii isolates from bats. A total of 369 bats from different municipalities in São Paulo state, southeastern Brazil, were captured and euthanised, and collected tissues (heart and pectoral muscle) were processed for each bat or in pools of two or three bats and bioassayed in mice (a total of 283 bioassays). Eleven PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) markers were used to genotype positive samples: SAG1, SAG2 (5'-3'SAG2 and alt. SAG2), SAG3, BTUB, GRA6, L358, c22-8, c29-2, PK1, CS3 and Apico. The parasite was isolated from two bats from São Paulo city: an insectivorous bat, the velvety free-tailed bat Molossus molossus, and a hematophagous bat, the common vampire bat Desmodus rotundus. Isolates were designated TgBatBr1 and TgBatBr2, respectively. The genotype of the isolate from M. molossus (TgBatBr1) has been previously described in an isolate from a capybara from São Paulo state, and the genotype from the D. rotundus isolate (TgBatBr2) has already been identified in isolates from cats, chickens, capybaras, sheep, a rodent and a common rabbit from different Brazilian states, suggesting that this may be a common T. gondii lineage circulating in some Brazilian regions. Isolation of T. gondii from a hematophagous species is striking. This study reveals that bats can share the same isolates that are found in domesticated and wild terrestrial animals. This is the first report of the isolation and genotyping of T. gondii in chiropterans.

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Neospora caninum, a cause of abortion and stillbirth in cattle, was studied by histology, immunohistochemistry, and nested-PCR, using primers from the Nc5 region of the genomic DNA (PCR PLUS) and primers from the ITS1 region of the ribosomal DNA (PCR JB). A total of 105 fetal samples sent to the Centro de Pesquisa e Desenvolvimento de Sanidade Animal do Instituto Biológico from January 2006 to May 2008 were examined for evidence of N. caninum. Histological examination revealed 71.4% with non-suppurative inflammation in the heart, lung, liver, kidney, placenta, and brain. Immunohistochemistry detected infections in 8.6% of the samples, mainly in the brain, placenta, and heart. Nested-PCR JB revealed 6.7% with infections, while nested-PCR PLUS returned 20.9% positive results, mainly in brain and placenta, and in the pooled liver and heart. Kappa statistics demonstrated little agreement among the three techniques. The three methods are complementary, since they have distinct diagnostic characteristics and were combined to give a positivity rate of 24.8%.

Brazil harbors the largest number of wild Neotropical felid species, with ten of the twelve species recorded in the American continent. Although these animals are considered to be definitive hosts for Toxoplasma gondii, there are few descriptions of the parasite in these species. Here, we performed a molecular detection of T. gondii by amplification of the marker ITS-1 from tissue samples obtained from 90 free-ranging wild small Neotropical felids from Rio Grande do Sul - Brazil. Of the sampled animals, 34.4% (n=31) were positive including the species Puma yagouaroundi - jaguarondi (9/22), Leopardus geoffroyi - Geoffroy's cat (6/22), Leopardus tigrinus - oncilla (8/28), Leopardus wiedii - margay (6/10), Leopardus pardalis - ocelot (1/1) and Leopardus colocolo - Pampas cat (1/7). Toxoplasma DNA was detected with a frequency of 14.6% (63/433) in primary samples of tongue (16/56), brain (8/43), skeletal muscle (15/83), heart (7/63), diaphragm (3/56), vitreous humor (2/44), eye muscle (6/44) and eyeball (6/44). Multilocus PCR-RFLP genotyping of eleven small Neotropical felids using the molecular markers SAG1, 5'3'SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3 allowed the partial characterization of eight genotypes. We fully characterized two new genotypes that have not been described previously in Brazil (Lw#31Tn from L. wiedii and Py#21Sm from P. yagouaroundi) and one genotype Py#56Br from P. yagouaroundi that has been described previously in isolates from cats, dogs and capybaras from São Paulo state. This study constitutes the first detection and genotypic characterization of T. gondii in free-ranging felids in Brazil, demonstrating the occurrence of the parasite in wild populations and suggesting its potential transmissibility to humans and other domestic and wild animals.

Zika virus (ZIKV), an emerging arthropod-borne virus (arbovirus) of the Flaviviridae family, is a current issue worldwide, particularly because of the congenital and neurological syndromes associated with infection by this virus. As the initial clinical symptoms of all diseases caused by this group are very similar, clinical diagnosis is difficult. Furthermore, laboratory diagnostic efforts have failed to identify specific and accurate tests for each virus of the Flaviviridae family due to the cross-reactivity of these viruses in serum samples. This situation has resulted in underreporting of the diseases caused by flaviviruses. However, many companies developed commercial diagnostic tests after the recent ZIKV outbreak. Moreover, health regulatory agencies have approved different commercial tests to extend the monitoring of ZIKV infections. Considering that a specific and sensitive diagnostic method for estimating risk and evaluating ZIKV propagation is still needed, this review aims to provide an update of the main commercially approved serological diagnostics test by the US Food and Drug Administration (FDA) and Brazilian National Health Surveillance Agency (ANVISA). Additionally, we present the technologies used for monoclonal antibody production as a tool for the development of diagnostic tests and applications of these antibodies in detecting ZIKV infections worldwide.