Although serological assays have been widely used for the diagnosis of canine visceral leishmaniasis (CVL), they present different performances depending on the clinical profile of the dogs. This study evaluated the accuracy of serological tests, immunochromatographic (Dual Path Platform: DPP®) and enzyme-linked immunosorbent (ELISA EIE®), for CVL in relation to the detection of Leishmania DNA through real-time polymerase chain reaction (real-time PCR) in samples from symptomatic and asymptomatic dogs from a non-endemic area in the state of Rio Grande do Sul, Southern Brazil. Serum from 140 dogs (39 symptomatic and 101 asymptomatic) was tested by DPP and ELISA followed by real-time PCR. From a total of 140 samples evaluated, Leishmania DNA was detected by real-time PCR in 41.4% (58/140). Moreover, 67.2% of samples positive in real-time PCR were positive in both DPP and ELISA (39/58), showing moderate agreement between methods. In the symptomatic group, one sample non-reactive in both serological assays was positive in real-time PCR, whereas in the asymptomatic group, 17.8% non-reactive or undetermined samples in serological assays were positive in the molecular method. Leishmania DNA was not detected in 17.9% reactive samples by serological assays from the symptomatic group, and in 3.9% from asymptomatic dogs. Real-time PCR demonstrated greater homogeneity between symptomatic and asymptomatic groups compared with DPP and ELISA. The molecular method can help to establish the correct CVL diagnosis, particularly in asymptomatic dogs, avoiding undesirable euthanasia.

Severe acute respiratorysyndrome coronavirus‐2 (SARS‐CoV‐2) pandemic spread rapidly and this scenario is concerning worldwide, presenting more than 590 million coronavirus disease 2019 cases and 6.4 million deaths. The emergence of novel lineages carrying several mutations in the spike protein has raised additional public health concerns worldwide during the pandemic. The present study review and summarizes the temporal spreading and molecular evolution of SARS‐CoV‐2 clades and variants worldwide. The evaluation of these data is important for understanding the evolutionary histories of SARSCoV‐2 lineages, allowing us to identify the origins of each lineage of this virus responsible for one of the biggest pandemics in history. A total of 2897 SARS‐CoV‐2 whole‐genome sequences with available information from the country and sampling date (December 2019 to August 2022), were obtained and were evaluated by Bayesian approach. The results demonstrated that the SARS‐CoV‐2 the time to the most recent common ancestor (tMRCA) in Asia was 2019‐12‐26 (highest posterior density 95% [HPD95%]: 2019‐12‐18; 2019‐12‐29), in Oceania 2020‐01‐24 (HPD95%: 2020‐01‐15; 2020‐01‐30), in Africa 2020‐02‐27 (HPD95%: 2020‐02‐21; 2020‐03‐04), in Europe 2020‐02‐27 (HPD95%: 2020‐02‐20; 2020‐03‐06), in North America 2020‐03‐12 (HPD95%: 2020‐03‐05; 2020‐03‐18), and in South America 2020‐03‐15 (HPD95%: 2020‐03‐09; 2020‐03‐28). Between December 2019 and June 2020, 11 clades were detected (20I [Alpha] and 19A, 19B, 20B, 20C, 20A, 20D, 20E [EU1], 20F, 20H [Beta]). From July to December 2020, 4 clades were identified (20J [Gamma, V3], 21 C [Epsilon], 21D [Eta], and 21G [Lambda]). Between January and June 2021, 3 clades of the Delta variant were detected (21A, 21I, and 21J). Between July and December 2021, two variants were detected, Delta (21A, 21I, and 21J) and Omicron (21K, 21L, 22B, and 22C). Between January and June 2022, the Delta (21I and 21J) and Omicron (21K, 21L, and 22A) variants were detected. Finally, between July and August 2022, 3 clades of Omicron were detected (22B, 22C, and 22D). Clade 19A was first detected in the SARS‐CoV‐2 pandemic (Wuhan strain) with origin in 2019‐12‐16 (HPD95%: 2019‐12‐15; 2019‐12‐25); 20I (Alpha) in 2020‐11‐24 (HPD95%: 2020‐11‐15; 2021‐12‐02); 20H (Beta) in 2020‐11‐25 (HPD95%: 2020‐11‐13; 2020‐11‐29); 20J (Gamma) was 2020‐12‐21 (HPD95%: 2020‐11‐05; 2021‐01‐15); 21A (Delta) in 2020‐09‐20 (HPD95%: 2020‐05‐17; 2021‐02‐03); 21J (Delta) in 2021‐02‐26 (2020‐11‐02; 2021‐04‐24); 21M (Omicron) in 2021‐01‐25 (HPD95%: 2020‐09‐16; 2021‐08‐08); 21K (Omicron) in 2021‐07‐30 (HPD95%: 2021‐05‐30; 2021‐10‐19); 21L (Omicron) in 2021‐10‐03 (HPD95%: 2021‐04‐16; 2021‐12‐23); 22B (Omicron) in 2022‐01‐25 (HPD95%: 2022‐01‐10; 2022‐02‐05); 21L in 2021‐12‐20 (HPD95%: 2021‐05‐16; 2021‐12‐31). Currently, the Omicron variant predominates worldwide, with the 21L clade branching into 3 (22A, 22B, and 22C). Phylogeographic data showed that Alpha variant originated in the United Kingdom, Beta in South Africa, Gamma in Brazil, Delta in I...

In Brazil, visceral leishmaniasis (VL) is expanding and becoming urbanized, especially in non-endemic areas such as the State of Rio Grande do Sul. Considering that infected dogs are the main reservoir for zoonotic VL, this study evaluated the prevalence of canine visceral leishmaniasis (CVL) in the metropolitan area of Porto Alegre, a new area of expansion of VL in Brazil. Serum and plasma from 405 asymptomatic dogs from the municipalities of Canoas (n=107), São Leopoldo (n=216), and Novo Hamburgo (n=82) were tested for CVL using immunochromatographic (DPP®) and ELISA EIE® assays (2 assays officially adopted by the Brazilian government for the diagnosis of CVL) and real-time PCR to confirm the results. There was no agreement among serological and real-time PCR results, indicating that the Leishmania infection in asymptomatic animals with low parasite load, confirmed by negative parasitological tests (smears and parasite culture), need to be evaluated by molecular methods. The prevalence of LVC in the metropolitan region of Porto Alegre, confirmed by real-time PCR was 4% (5.6% in Canoas and 4.6% in São Leopoldo). The use of molecular method is essential for accurate diagnosis of CVL, especially in asymptomatic dogs in non-endemic areas.

We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. Several solutions were evaluated as to their ability to liquefy a mucin-based matrix. Each liquefied matrix, supplemented with either Mycobacterium tuberculosis (MTB) H37Rv strain DNA or intact cells, was aliquoted onto a filter paper embedded with solubilizing agents, and was subsequently dried up. Most of the nucleic acids, including genomic DNA from the bacilli and the host, binds to the filter paper. Next, several protocols were evaluated to elute the DNA from the paper, using qPCR to detect the insertion sequence IS6110, a M. tuberculosis complex genomic marker. The limit of detection (LOD) of the best protocol was then evaluated using parallel seeding and colony counting. The protocol was also evaluated using seventeen sputum samples, previously characterized by the GeneXpert or culture. Two instruments (the ABI7500 Standard and the Q3-Plus system) and two reagents storage formats (frozen or ready-to-use) were evaluated. Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95°C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof–of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings.