Seroprevalence of antibodies against SARS-CoV-2 in the school community in Campo Grande, state of Mato Grosso do Sul, Brazil, October 2021-November 2022.

Introduction: With the reopening of schools during the coronavirus disease 2019 (COVID-19) pandemic, it was imperative to understand the role of students and education professionals in the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we determined the seroprevalence of the SARS-CoV-2 anti-nucleocapsid antibodies in the school community in Campo Grande, the capital and most populous city of the state of Mato Grosso do Sul (Brazil) and evaluated its association with sex, school level, and school type. Materials and methods: The survey was carried out in 20 public and private schools in the urban region of Campo Grande using the TR DPP® COVID-19 immunoglobulin M/immunoglobulin G (IgM/IgG) kit from the Immunobiological Technology Institute (Bio-Manguinhos, Rio de Janeiro, Brazil). Testing was carried out in three periods: from October to December 2021; from March to July 2022; and from August to November 2022. The participants were students aged 6-17 years enrolled in primary or secondary schools and professionals of different ages and roles. Results: During the first testing period, 162 participants were seropositive for the IgM and/or IgG anti-nucleocapsid SARS-CoV-2 antibodies, with an estimated seroprevalence of 19.6% using Bayesian multilevel regression. In the second period, 251 participants were seropositive (estimated seroprevalence, 34.6%), while in the third period, 393 participants were seroconverted (estimated seroprevalence, 56.7%). In 2022, there was an increase in the seroconversion rate compared to that in 2021. The most frequently described acute manifestations in the three periods were fever, headache, sore throat, and runny nose. In terms of the demographic profile, there was no predominance of seropositivity between the sexes, although women represented approximately 70% of the study population. There were also no differences between students and school staff. Discussion: The results made it possible to evaluate the extent of SARS-CoV-2 transmission in the school community through immunity developed against the virus, in addition to providing information about COVID-19 symptoms in children, adolescents, and adults.

Development and validity assessment of ELISA test with recombinant chimeric protein of SARS-CoV-2.

Serological tests developed for COVID-19 diagnostic are based on antibodies specific for SARS-CoV-2 antigens. Most of the antigens consist of a fragment or a whole amino acid sequence of the nucleocapsid or spike proteins. We evaluated a chimeric recombinant protein as an antigen in an ELISA test, using the most conserved and hydrophilic portions of the S1-subunit of the S and Nucleocapsid (N) proteins. These proteins, individually, indicated a suitable sensitivity of 93.6 and 100% and a specificity of 94.5 and 91.3%, respectively. However, our study with the chimera containing S1 and N proteins of SARS-CoV-2 suggested that the recombinant protein could better balance both the sensitivity (95.7%) and the specificity (95.5%) of the serological assay when comparing with the ELISA test using the antigens N and S1, individually. Accordingly, the chimera showed a high area under the ROC curve of 0.98 (CI 95% 0.958-1). Thus, our chimeric approach could be used to assess the natural exposure against SARS-CoV-2 virus over time, however, other tests will be necessary to better understand the behaviour of the chimera in samples from people with different vaccination doses and/or infected with different variants of the virus.

Evaluation of Molecular Methods to Identify Chagas Disease and Leishmaniasis in Blood Donation Candidates in Two Brazilian Centers.

In Brazil, blood donation is regulated by the Brazilian Ministry of Health, and all States follow the same protocol for clinical and laboratory screening. Brazil is an endemic country for Chagas disease (CD), caused by Trypanosoma cruzi, and for leishmaniasis, caused by a species of Leishmania spp. Screening for leishmaniosis is not routinely performed by blood banks. Given the antigenic similarity between T. cruzi and Leishmania spp., cross-reactions in serological tests can occur, and inconclusive results for CD have been found. The objective of this study was to apply molecular techniques, e.g., nPCR, PCR, and qPCR, to clarify cases of blood donation candidates with non-negative serology for CD and to analyze the difference between the melting temperature during real-time PCR using SYBR Green. Thirty-seven cases that showed non-negative results for CD using chemiluminescent microparticle immunoassay (CMIA) tests from blood banks in Campo Grande, MS, and Campinas, SP, were analyzed. In the serum samples, 35 samples were evaluated by ELISA, and 24.3% (9/35) showed positive results for CD. nPCR was able to detect 12 positive results in 35 samples (34.28%). qPCR for T. cruzi was quantifiable in the samples that showed a value ≥0.002 par eq/mL (parasite equivalents per milliliter), and in 35 samples, 11 (31.42%) were positive. Of all evaluated samples using the described tests (CMIA, ELISA, nPCR, and qPCR), 18 (48.6%) were positive for CD. For MCA by qPCR, the melting temperature was 82.06 °C ± 0.46 for T. cruzi and 81.9 °C ± 0.24 for Leishmania infantum. The Mann-Whitney test showed a significant value of p < 0.0001. However, the differentiation between T. cruzi and L. infantum could not be considered due to temperature overlap. For leishmaniasis, of the 35 samples with non-negative serology for CD tested by the indirect fluorescent antibody test (IFAT), only one sample (2.85%) was positive (1:80). The PCR for Leishmania spp. was performed on 36 blood samples from donation candidates, and all were negative. qPCR for L. infantum showed 37 negative results for the 37 analyzed samples. The data presented here show the importance of performing two different tests in CD screening at blood banks. Molecular tests should be used for confirmation, thereby improving the blood donation system.

Parasite load evaluation by qPCR and blood culture in Chagas disease and HIV co-infected patients under antiretroviral therapy.

Chagas disease also known as American trypanosomiasis, is caused by Trypanosoma cruzi and transmitted by triatominae-contaminated feces. It is considered a neglected tropical disease that affects 6 to 7 million people worldwide. The reactivation of Chagas disease occurs when the chronically infected hosts are not able to control T. cruzi infection, generating recurrence of the acute phase. HIV is the main immunosuppressive infection that can lead to the reactivation of chronic Chagas disease in AIDS conditions. In co-infected patients, the reactivation of Chagas disease is related to their high parasite load, high HIV viral load, and CD4 T-cell counting less than 200/mm3, which may evolve to meningoencephalitis and myocarditis. Eight T. cruzi/HIV co-infected patients under antiretroviral therapy (ART) and ten Chagas disease patients without HIV infection that attended at Study Group of Chagas Disease, Hospital de Clínicas, University of Campinas (GEdoCh/HC/UNICAMP-SP) and Pontifical Catholic University of Campinas SP (PUCC/SP) were evaluated. Tests for Chagas disease were performed, such as qPCR and T. cruzi blood culture. The patient's medical records were analyzed to verify clinical and epidemiological data, viral load, and CD4 T-cell counting since the outset of ART. For both groups, we found no statically significant differences between parasite load via blood culture and qPCR. In T. cruzi/HIV co-infected subjects, we observed a significant increase of CD4 T-cells counting and viral load decrease, which became undetectable over the years after ART. Parasites isolated from the patient's blood culture were genotyped, being the majority of them infected with TcII and one case of mixed infection (TcII and TcV/TcVI). These results were expected according to the region of origin of the patients. We suggest that the parasite load be monitored through qPCR in T.cruzi/HIV co-infected patients. We conclude that ART in people living with HIV improves infection and immunosuppression control, enabling the natural evolution of the American trypanosomiasis.

Neonatal sepsis: evaluation of risk factors and histopathological examination of placentas after delivery / Sepse neonatal: avaliação dos fatores de risco e análise histopatológica de placentas após o parto

Neonatal sepsis is a clinical syndrome defined by systemic signs of infection in newborns accompanied by bacteremia. Can be responsible for serious consequences for the newborn child,characterized at the birth as early sepsis or late onset sepsis, with high rate of neonatal morbidity and mortality. Pathological agents such as Escherichia coli (E. coli), Streptococcus agalactiae (S. agalactiae), Ureaplasma urealyticum and Mycoplasma hominis are most often responsible for intrauterine infections. The objective of this study is to evaluate the factors of neonatal sepsis predisposition in pregnant women through histopathological examination and the apoptotic index of placental tissues and detect DNA of E. coli and S. agalactiae using the Polymerase Chain Reaction (PCR). Histopathological analyses were made and the apoptotic index was determined to verify the levels of possible inflammatory infiltrates and cell death. Placenta samples were collected from November 2013 to May 2014. After DNA extraction, a PCR was performed amplifying the target fragment from the conserved regions of the rpoB (beta-RNA polymerase) polymorphism of E. coli and the factor 1 of S. agalactiae. The apoptosis index was tested with Acridine Orange and the histological procedure with Hematoxylin-Eosin staining. Among 100 samples of placental tissues analyzed by PCR, 48 represented the control group and did not present a risk factor associated with neonatal sepsis, and 52 samples representing the study group had at least one risk factor. Among these 52 samples, 7 (13.4%) had a PCR positive for E. coli. No placenta samples showed a positive PCR for S. agalactiae. The quantification of the apoptotic index did not show statistical significances between the groups and no inflammatory infiltrates were observed. However, histological sections showed fibrinoid necrosis, infarct areas and areas of calcification in all samples. Therefore, the results allow to conclude that the seven patients of experimental group with positive PCR for E. coli had eminent risk factors of neonatal sepsis, and the infection of the urinary tract (UTI) is the main aggravating circumstance. The histopathological examination demonstrated that the risk factors caused significant alterations, producing fibrinoid necrosis and infarcted areas in the placenta, contrary to apoptotic index that didn't differ from the group with unprecedented risk

A sepse neonatal é uma síndrome clínica definida por sinais sistêmicos de infecção em recém-nascidos acompanhados de bacteremia. Pode ser responsável por sérias consequências para o recém-nascido, caracterizadas ao nascimento como sepse precoce ou sepse tardia, com alta taxa de morbidade emortalidade neonatal. Agentes patológicos como Escherichia coli (E. coli), Streptococcus agalactiae (S. agalactiae), Ureaplasma urealyticum e Mycoplasma hominis são mais frequentemente responsáveis por infecções intra-uterinas. O objetivo deste estudo é avaliar os fatores de predisposição da sepse neonatal em gestantes através do exame histopatológico e do índice apoptótico de tecidos placentários e detectar DNA de E. coli e S. agalactiae utilizando a reação em cadeia da polimerase (PCR). Análises histopatológicas foram realizadas e o índice apoptótico foi determinado para verificar os níveis de possíveis infiltrados inflamatórios e morte celular. Amostras de placenta foram coletadas de novembro de 2013 a maio de 2014. Após a extração de DNA, foi realizada uma PCR amplificando o fragmento alvo das regiões conservadas do polimorfismo rpoB (polimerase beta-RNA) de E. coli e o fator 1 de S. agalactiae. O índice de apoptose foi testado com alaranjado de acridina e o procedimento histológico com coloração de hematoxilina-eosina. Entre 100 amostras de tecidos placentários analisados por PCR, 48 representaram o grupo controle e não apresentaram fator de risco associado à sepse neonatal, e 52 amostras representativas do grupo de estudo apresentaram pelo menos um fator de risco. Entre essas 52 amostras, 7 (13,4%) apresentaram PCR positivo para E. coli. Nenhuma amostra de placenta foi positivo para S. agalactiae na PCR. A quantificação do índice apoptótico não mostrou significância estatística entre os grupos e não foram encontrados infiltrados inflamatórios. No entanto, cortes histológicos mostraram necrose fibrinóide, áreas de infarto e áreas de calcificação em todas as amostras. Portanto, os resultados permitem concluir que as sete pacientes do grupo experimental com PCR positivo para E. coliapresentavam fatores de risco eminentes de sepse neonatal, sendo a infecção do trato urinário (ITU), o principal agravante. O exame histopatológico demonstrou que os fatores de risco causaram alterações significativas, produzindo necrose fibrinóide e áreas infartadas na placenta, ao contrário o índice apoptótico que não diferiu do grupo sem precedentes de risco.

Serological and molecular inquiry of Chagas disease in an Afro-descendant settlement in Mato Grosso do Sul State, Brazil.

Furnas do Dionísio is a Brazilian Afro-descendant settlement in the city of Jaraguari, 21.4 miles from Campo Grande, Mato Grosso do Sul, Brazil. Approximately 96 families live in this quilombola (Maroon) settlement, also known in Brazil as a remnant community of descendants of African slaves. Recent studies found 20% of households were infested by triatomines, 18% of insects captured in the community were infected by Trypanosoma cruzi, and 22.7% of dogs presented T. cruzi antibodies. The low prevalence of Chagas disease observed in humans in Mato Grosso do Sul State is attributed to its arrival via colonist migration and subsequent transplacental transmission. In order to gain a better understanding of the T. cruzi cycle in residents of the study community, serological and molecular tests were carried out to diagnose Chagas disease. In the present study, 175 residents between 2 and 80 years old were included. A total of 175 participants were interviewed and 170 provided blood samples, which were tested for T. cruzi antibodies with serological tests. Molecular diagnosis was performed in 167 participants by PCR (KDNA) and NPCR (satellite DNA) tests. One of the 170 samples tested positive for all serological tests performed. The overall frequency of Chagas disease in the community was low (0.6%). Interview responses revealed that 66.3% knew of triatomine insects and 65.7% reported having had no contact with them. Physical improvements to residences, together with vector surveillance and control by the State and municipal governments and local ecological conservation contribute to the low frequency of the Chagas disease in this quilombola community.

Leishmania (V.) braziliensis infecting bats from Pantanal wetland, Brazil: First records for Platyrrhinus lineatus and Artibeus planirostris.

In the New World genus Leishmania parasites are etiological agents of neglected zoonoses known as leishmaniasis. Its epidemiology is very complex due to the participation of several species of sand fly vectors and mammalian hosts, and man is an accidental host. Control is very difficult because of the different epidemiological patterns of transmission observed. Studies about Leishmania spp. infection in bats are so scarce, which represents a large gap in knowledge about the role of these animals in the transmission cycle of these pathogens, especially when considering that Chiroptera is one of the most abundant and diverse orders among mammals. Leishmaniasis in Mato Grosso do Sul, Brazil are remarkably frequent, probably due to the abundance of its regional mastofauna. The recent record of L. braziliensis in bats from this state indicates the need to clarify the role of these mammals in the transmission cycle. In this study we evaluated the presence of Leishmania parasites in the skin of different species of bats, using PCR directed to Leishmania spp. kDNA for screening followed by PCR/RFLP analysis of the hsp70 gene for the identification of parasite species. Leishmania species identification was confirmed by PCR directed to the G6PD gene of L. braziliensis, followed by sequencing of the PCR product. Samples from 47 bats were processed, of which in three specimens (6.38%) was detected the presence of Leishmania sp. kDNA. PCR/RFLP and sequencing identified the species involved in the infection as L. braziliensis in all of them. This is the first report of Leishmania braziliensis in bats from Pantanal ecosystem and the first record of this species in Platyrrhinus lineatus and Artibeus planirostris, bats with a wide distribution in South America. These results reinforce the need to deepen the knowledge about the possibility of bats act as reservoirs of Leishmania spp. especially considering their ability of dispersion and occupation of anthropic environments.

The detection of Trypanosoma cruzi by nested-PCR in elderly patients: relationship to the clinical and epidemiological profile.

Chagas disease, which is caused by Trypanosoma cruzi, is transmitted primarily by triatomine bugs, although the incidence of new cases has decreased as a result of vector control. In Brazil, most of those affected have the chronic form of the disease and are generally elderly individuals who require appropriate clinical follow-up. In this work, we undertook a descriptive study in which 85 patients were interviewed and blood samples were collected for molecular analyses based on the amplification of parasite satellite DNA. The cardiac form of the disease was the most prevalent among the patients and hypertension was the most frequent comorbidity; polypharmacy was detected in 34% of the cases. Serological tests were positive in 95% of cases while 36% were positive in nested-polymerase chain reaction. These findings indicate an increased use of medications and a larger number of age-related diseases in elderly patients with Chagas disease, even in patients with low parasitemia. We conclude that elderly patients with Chagas disease require special attention and that further studies should be done with elderly individuals who carry this disease.

Use of a nested polymerase chain reaction (N-PCR) to detect Trypanosoma cruzi in blood samples from chronic chagasic patients and patients with doubtful serologies.

Chagas disease, caused by Trypanosoma cruzi, is an important endemic illness in Latin America. Serologic tests for T. cruzi detection in blood are sensitive, but their specificity is unsatisfactory. Direct detection of parasites in blood, either by xenodiagnosis or hemoculture, is highly specific but of low sensitivity. Molecular assays such as the Polymerase chain reaction (PCR), which amplifies certain repetitive sequences of nuclear DNA has been used as a good alternative tool for T. cruzi detection in human blood. The present study aimed to test PCR diagnosis in chagasic chronic patients and doubtful serologic patients attended in GEDOCH (Chagas Disease Study Group/UNICAMP, Brazil). A 149 bp fragment originated from nuclear DNA was specifically detected in chronic chagasic patients. The results of these tests were compared with serologic diagnosis performed using standard techniques and xenodiagnosis. We found that 43 out of 50 patients previously serodiagnosed as chagasic were positive using the N-PCR method. Thirteen of 30 patients with doubtful serologic results were confirmed as positive by N-PCR. Our results suggest that the N-PCR may be a complementary tool to serology in the diagnosis of Chagas disease, and that it is usefull for parasite detection in patients with chronic disease and patients with doubtful serologic results.