SARS-CoV-2 testing among patients and healthcare professionals in an HIV outpatient clinic in Brazil.

The COVID-19 pandemic in Brazil has been marked by high infection and death rates. The immune response generated by current vaccination might be influenced by previous natural infection, and baseline estimates may help in the evaluation of vaccine-induced serological response. We evaluated previous SARS-CoV-2 testing (RT-PCR), and performed rapid diagnostic tests (RDT) and high throughput electrochemiluminescence immunoassay (ECLIA) before vaccination among people living with HIV (PLWH), users of antiretroviral prophylaxis (PrEP/PEP), and healthcare professionals in an HIV outpatient clinic (HCP-HC). RDT was positive in 25.7% (95% CI: 19-33%) overall, 31.3% (95% CI : 18-45%) among PLWH, 23.7% (95% CI : 14-34%) in PrEP/PEP users and 21.4% (95% CI : 05-28%) in HCP-HC (p=0.548). Diagnostic RT-PCR testing was very limited, even for symptomatic individuals, and whereas all HCP-HC had one test perfomed, only 35% of the patients (PREP/PEP/PLWH) were tested (p<0.0001). Adequate monitoring of post-vaccination humoral response and breakthrough infections including those in asymptomatic cases are warranted, especially in immunologically compromised individuals.

A simple algorithm for selecting cases to investigate acute and early HIV infections in low- and middle-income countries.

We documented the outcome of an over 10-year (2011-2021) effort to diagnose acute and early HIV infections (AEHI) in an Infectious Diseases Outpatient Clinic with limited resources. Of a total of 132, 119 HIV-RNA tests were performed from 2017 to 2020, 12 cases were identified, using a simple algorithm: risk exposure of 6 weeks or less before the visit and/or symptoms compatible with acute retroviral syndrome 7-30 days after exposure and/or undetermined 3rd generation rapid diagnostic test or serology. AEHI diagnoses varied from 2.4% among asymptomatic to 25% for undetermined serology cases using this simple screening applicable to different settings.

SARS-CoV-2 testing among patients and healthcare professionals in an HIV outpatient clinic in Brazil

ABSTRACT The COVID-19 pandemic in Brazil has been marked by high infection and death rates. The immune response generated by current vaccination might be influenced by previous natural infection, and baseline estimates may help in the evaluation of vaccine-induced serological response. We evaluated previous SARS-CoV-2 testing (RT-PCR), and performed rapid diagnostic tests (RDT) and high throughput electrochemiluminescence immunoassay (ECLIA) before vaccination among people living with HIV (PLWH), users of antiretroviral prophylaxis (PrEP/PEP), and healthcare professionals in an HIV outpatient clinic (HCP-HC). RDT was positive in 25.7% (95% CI: 19-33%) overall, 31.3% (95% CI : 18-45%) among PLWH, 23.7% (95% CI : 14-34%) in PrEP/PEP users and 21.4% (95% CI : 05-28%) in HCP-HC (p=0.548). Diagnostic RT-PCR testing was very limited, even for symptomatic individuals, and whereas all HCP-HC had one test perfomed, only 35% of the patients (PREP/PEP/PLWH) were tested (p<0.0001). Adequate monitoring of post-vaccination humoral response and breakthrough infections including those in asymptomatic cases are warranted, especially in immunologically compromised individuals.

Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine (CoronaVac) in inadvertently vaccinated healthy children.

Twenty-seven children aged seven months to 5 years were inadvertently vaccinated with a COVID-19 vaccine, the CoronaVac (Sinovac, China), an inactivated SARS-CoV-2 vaccine, in two different cities of Sao Paulo State, Brazil. After the event, these children were monitored by local pediatricians and serum samples were collected at the first visit and 30 days after vaccination and tested for SARS-CoV-2 S1 serology with Ortho total IgG anti-S1 protein and Cpass, an ACE2 receptor binding domain inhibition assay. Only one child had a mild symptom after vaccination, with no other adverse events documented up to the 30 days follow-up. Of 27 children tested 3-9 days after vaccination, 5 (19%) had positive serology suggesting a previous natural SARS-CoV-2 infection, with all 19 tested on day 30 after vaccination and presenting with positive tests, with an increment of antibody titers in those initially positive. A low Cpass binding inhibition was observed in the first collection in 11 seronegative cases, with high titers among those anti-S1 positive. All children showed an important increase in antibody titers on day 30. The event allowed the documentation of a robust serological response to one dose of CoronaVac in this small population of young children, with no major adverse effects. Although it was an unfortunate accident, this event may contribute with future vaccine strategies in this age group. The data suggest that CoronaVac is safe and immunogenic for children.

Extracellular vesicles isolated from Toxoplasma gondii induce host immune response.

This study established a protocol to purify Toxoplasma gondii tachyzoite microvesicles and exosomes, called as extracellular vesicles (EVs). In addition, the investigations were conducted to determine the kinetic of EV release by tachyzoites and whether EV proteins are able to modulate the host immune response. The particle size and concentration released by tachyzoites in culture medium at different incubation-period were characterized by nanoparticle tracking analysis. Tachyzoites (1 × 106 ) released around 4.37 ± 0.81 × 108 EVs/mL/h, with size varying between 138.2 and 171.9 nm. EVs released into the medium were purified by gel-exclusion chromatography and screened by ELISA, using a pool of human positive sera for toxoplasmosis. EV-fractions contained high concentration of proteins, and EVs were analyzed by scanning and transmission electron microscopies. Tachyzoites released EVs into the culture medium throughout all membrane surface, and these vesicles contain small RNAs/miRNA. Pooled sera from chronically infected human or mice (infected with 2 different T. gondii strains) recognized distinct EV electrophoretic patterns in immunoblotting. T. gondii EVs significantly induced IL-10, TNF-α and iNOS in murine macrophages. In conclusion, this study shows that T. gondii secrete/excrete EVs (microvesicles and exosomes) contain miRNA and they were immunologically recognized by host immune response.

Purificação e caracterização de vesículas extracelulares de taquizoítos de toxoplasma gondii / Purification and characterization of extracellular vesicles of taquizoítos of toxoplasma gondii

Toxoplasma gondii, protozoário causador da toxoplasmose, é transmitido dos animais para os seres humanos pela ingestão de carne contaminada ou por oocistos liberados nas fezes de felinos no ambiente. Nos seres humanos, a infecção é normalmente assintomática, mas em casos em que a infecção primária ocorre durante a gravidez ou a reativação da infecção latente ocorre em pacientes imunosuprimidos pode ser grave. Nas últimas décadas tem se estudado pequenas estruturas secretadas pelas células procarióticas e eucarióticas denominadas de vesículas extracelulares (EVs). As EVs podem transportar biomarcadores de doenças, macromoléculas biorreativas contribuindo para a patogênese e sendo uma forma de diagnóstico não invasivo. Estas pequenas estruturas podem ser isoladas por ultracentrifugação, cromatografia e observadas por microscopia eletrônica. Elas participam na comunicação entre as células, na transferência de proteínas, lipídios e ácidos nucléicos. Diante do exposto, o presente estudo teve como objetivo estabelecer um protocolo para isolar e caracterizar vesículas extracelulares produzidas e excretadas por taquizoítos da cepa RH de T. gondii. Taquizoítos provenientes de culturas de células VERO foram isolados dos sobrenadantes das culturas e centrifugados em cinco séries de lavagens. A seguir, foram incubados em meio de cultura por 24 horas para secreção das EVs. Em seguida, investigou-se o tamanho e concentração das EVs isoladas por análise de varredura de partículas (NTA) no equipamento NanoSight. Paralelamente, a morfologia e a liberação das EVs também foram investigadas por microscopia eletrônica de transmissão e de varredura. Então, as EVs foram purificadas por cromatografia em gelexclusão em alíquotas de 1 mL (24-32 frações) e imuno-selecionadas por ELISA utilizando um "pool" de soros reagente para toxoplasmose. A seguir foi investigado a presença de miRNA nas EVs; e finalmente, foi avaliado o perfil proteico das EVs de T. gondii para verificar por testes sorológicos se estas partículas poderiam ser reconhecidas pelo sistema imune hospedeiro. As análises realizadas por NTA permitiram determinar que cerca de 1 x 106 taquizoítos secretaram de 4 a 8 x 108 EVs/mL num período de 24 horas de incubação em meio de cultura celular. Adicionalmente, estas vesículas apresentaram morfologia e tamanho de 165-175 nm de diâmetro, tamanho correspondente às microvesículas. Estes resultados também foram confirmados pela avaliação das imagens fornecidas pelas microscopias eletrônicas de transmissão e varredura. Tambem foi possível determinar a presenção de small RNAs e micro RNAs através de uma corrida eletroforética micro fluídica. As análises por SDS-PAGE mostram que as proteínas que compõem as EVs apresentaram um perfil eletroforético com espectro de 15 a 70 kDa. Soros de camundongos cronicamente infectados (com 2 diferentes cepas de T. gondii) e soros humanos reconheceram distintos padrões eletroforéticos no immunoblotting. As vesículas liberadas por T. gondii podem ser um mecanismo importante pelo qual os parasitas apresentam seus antígenos ao hospedeiro e, portanto, podem ter um papel importante na patogênese da toxoplasmose

Toxoplasma gondii, a protozoan that causes toxoplasmosis, is transmitted from animals to humans through ingestion of infected meat or oocysts released by felines into the environment. In humans, infection is usually asymptomatic, but in cases where primary infection occurs during pregnancy or the reactivation of latent infection occurs in immunosuppressed patients it can be serious. In the last decades, small structures secreted by prokaryotic and eukaryotic cells called extracellular vesicles (EVs) were studied. These small structures can be isolated by ultracentrifugation, chromatography and examined by electron microscopy. EVs participate in the communication between cells, transfer of proteins, lipids and nucleic acids. In addition, them can carry disease biomarkers, bioreactive macromolecules contributing to the pathogenesis of diseases. In view of the above, the present study aimed to establish a protocol to isolate and characterize extracellular vesicles produced and excreted by tachyzoites of T. gondii RH strain. To achieve this goal, tachyzoites from VERO cell cultures were isolated from the culture supernatants and centrifuged in five sets of washes. Next, they were incubated in culture medium for 24 hours for secretion of the EVs. The size and concentration of the isolated EVs by particle scan analysis (NTA) were investigated on the NanoSight (NTA) equipment. In parallel, the morphology and the release of the EVs were also investigated by transmission and scanning electron microscopy. Then, EVs were purified by gel-exclusion chromatography in 1 mL aliquots (24-32 fractions) and immuno-selected by ELISA using a pool of reagent sera for toxoplasmosis. Next, the presence of miRNA in the EVs was investigated; and finally, the protein profile of T. gondii EVs was evaluated and verify by serological tests if these particles could be recognized by the host immune system. The results showed that the protocol for recovery of T. gondii EVs was established from 1 to 1010 tachyzoites obtained from cultured cells. Analyzes performed by NTA allowed us to determine that about 1 x 106 tachyzoites secreted 4 to 8 x 108 EVs / mL within 24 hours of incubation in culture medium. Additionally, these vesicles presented morphology and size of 165-175 nm of diameter, corresponding microvesicles size. These results were also confirmed by the evaluation of images provided by transmission and scanning electron microscopy. The miRNA purifications from the EVs and subsequent microfluidic electrophoretic run confirmed the presence of smallRNA and miRNA in the vesicles. SDS-PAGE analyzes show that the proteins carried by the EVs showed an electrophoretic profile with a spectrum of 15 to 70 kDa. Sera from chronically infected mice (with 2 different strains of T. gondii) and humam serum recognized distinct electrophoretic patterns in immunoblotting