Estabelecimento de um protocolo de Nested-PCR para detecção do vírus da anemia das galinhas e análise filogenética de cepas brasileiras

O vírus da anemia das galinhas (CAV) pode causar imunodepressão em galinhas de todas as idades e doença nos frangos jovens, a qual é caracterizada por severa anemia, atrofia da medula óssea e hemorragias. A doença clínica é rara hoje, porém a forma subclínica é, freqüentemente, encontrada em criações comerciais e resulta num considerável decréscimo do desempenho. O CAV apresenta variabilidade genética, entretanto, em relação às amostras brasileiras do CAV, pouco ou quase nada desta variabilidade é conhecida. O presente trabalho descreve um protocolo de Nested-PCR para a detecção do CAV diretamente de amostras clínicas e analisa filogeneticamente as seqüências nucleotídicas das amostras positivas buscando verificar se a patologia apresentada por estas está relacionada com a variabilidade genética. Para a extração de DNA, o método baseado em tiocianato de guanidina mostrou-se mais eficiente e prático de executar que os demais testados. Foi selecionado um par de primers que amplifica uma região de 664 pb do gene vp1 e outro par que amplifica uma região interna de 539 pb para a realização da Nested-PCR. A especificidade dos primers foi avaliada utilizando amostras de lotes controlados para CAV e 30 diferentes isolados de vírus e bactérias causadoras de doenças em galinhas, as quais não geraram produto de amplificação. A sensibilidade foi determinada a partir de diluições seriadas da vacina comercial para o CAV. A Nested-PCR mostrou ser mais sensível do que a PCR e foi capaz de detectar 0,16 DICC50% da cepa vacinal. Além disso, a Nested-PCR detectou DNA viral em tecidos, soro e cama aviária de lotes com e sem sintomas clínicos. O produto de amplificação de 539 pb do gene vp1 de 44 amostras, provenientes de diferentes Estados produtores de frangos do Brasil, foi seqüenciado e foram encontradas 10 novas seqüências nucleotídicas do CAV. Estas 10 seqüências nucleotídicas foram analisadas filogeneticamente pelo método de distância neighbour joining com 1000 replicações o qual, mostrou que não houve correlação entre a patogenia apresentada nos animais e os grupos genéticos. Estas seqüências nucleotídicas também foram comparadas com 30 cepas de CAV isoladas em outros países e não foi observada correlação entre a distribuição geográfica e a variabilidade genética. Substituições de amino ácidos foram observadas em 9 posições sendo que, 65R substituindo o resíduo Q e 98F substituindo o resíduo Y ainda não haviam sido observadas. Conclui-se que, como técnica de detecção do CAV, o protocolo de Nested-PCR aqui descrito é mais sensível e menos trabalhoso do que o isolamento viral. As amostras Brasileiras de CAV possuem características filogenéticas similares às isoladas em outros países

An improved method for characterization of the mutation associated to porcine stress syndrome by PCR amplification followed by restriction analysis

A mutation in the gene coding for the ryanodine receptor 1 (RYR1), also known as halothane (hal) gene or swine stress gene, is associated to the porcine stress syndrome (PSS). Detection of the mutation is normally accomplished by PCR amplification of an 81bp fragment of the hal gene, followed by digestion with the HhaI restriction endonuclease. Wild-type allele (N) is cut in two fragments, whereas the mutant allele (n) is not digested by the restriction enzyme. Electrophoresis of the digested DNA on agarose gel and ethidium bromide staining allows the reading of the result. The correct interpretation is difficult due to the small size of the DNA fragments. In this study we designed a new set of primers for amplification of a 144bp fragment that facilitates the reading of the result. In addition, we optimized the PCR reaction to allow amplification from a single hair bulb, added directly into the PCR mix without previous treatment. This improved method was used to genotype 165 sows and boars used in a breeding program. Forty-nine percent of the animals had the NN genotype, whereas 50% were Nn and only 1% was nn

NEUROSYPHILIS IN PEDIATRIC POPULATION: A REVIEW OF CASES

Background: Neurosyphilis is the most severe presentation of acquired and congenital syphilis, it occurs when Treponema pallidum penetrates the central nervous system. This development can occur at any time during the evolution of the disease phases. In Brazil in 2021, 2,343 children born with congenital syphilis were diagnosed with neurosyphilis, representing 9.3% of the total. The objective of this study was to analyze the scientific evidence on the medical manifestations presented by pediatric patients affected by neurosyphilis. Methods: In February 2023, PubMed, VHL, Scopus, Lilacs and Bdenf databases were researched for published case reports of patients aged zero to less than 18 years with T. pallidum infection. The data analysis period covered 55 years. Results: Nine articles were found in the period of publication from 1967 to 2022. There were two cases in females and only one with manifestation in an adolescent of acquired neurosyphilis. The adolescent's case was the only one with a favorable outcome after adequate treatment. Other cases resulted in impaired quality of life for patients and family members involved in child care. Conclusion: The reported cases of neurosyphilis were mostly related to late diagnosis. Evidencing the fragility of prenatal care, which is crucial for prevention and intervention by T. pallidum infection causes serious consequences for child development. Therefore, control measures should focus on mandatory prenatal screening during the first trimester of pregnancy, partner notification, prompt treatment and postnatal follow-up of the newborn

Neurosyphilis in Brazilian newborns: a health problem that could be avoided

Syphilis is a public health problem, especially in pregnant women, due to the risk of transmission to the fetus and the involvement of the central nervous system, causing neurosyphilis. A case-control study was carried out to analyze the variables associated with neurosyphilis in Brazilian newborns of pregnant women with syphilis admitted for childbirth. Newborns were submitted to treponemal and non-treponemal tests, cerebrospinal fluid analysis, and long bone radiography. Newborns diagnosed with neurosyphilis and congenital syphilis were defined as cases and controls, respectively. The length of hospitalization and mean cost of neurosyphilis treatment were also evaluated.Twenty-one cases of newborns with neurosyphilis and 42 controls with congenital syphilis were included in the study. Out of 63 pregnant women with syphilis, 95.2% (60/63) received prenatal care, 74.6% (47/63) were diagnosed with syphilis during this period, 31.9% (15/47) underwent treponemic tests, 80.8% (38/47) were treated with penicillin and only 46.8% (22/47) of the partners received the treatment. Clinical complications such as low birth weight were observed in 12.7% (8/63) of the newborns. About 50.8% (32/63) of the newborns were hospitalized due to syphilis complications and each case of neurosyphilis spent at least US$ 881.48 on treatment and hospitalization. The results showed that the prenatal coverage is not sufficient to prevent neurosyphilis. The late diagnosis of syphilis in pregnant women and inadequate follow-up of sexual partners may favor the vertical transmission of T. pallidum in pregnant Brazilian women. Thus, improving the quality of health services is important for a more effective control of neurosyphilis

Carbapenem-resistant Pseudomonas aeruginosa strains: a worrying health problem in intensive care units

Pseudomonas aeruginosa is one of the most common bacterium with a broad spectrum of human-associated infections. It is intrinsically resistant to many antimicrobial drugs, making carbapenems crucial in clinical management. The emergence and dissemination of carbapenemases among P. aeruginosa clinical isolates is a serious public health concern as it limits the options for the treatment of bacterial infections. Here, we described the molecular and epidemiological characteristics of 28 carbapenem-resistant P. aeruginosa strains isolated from patients hospitalized in an intensive care unit (ICU). The antimicrobial susceptibility of carbapenem-resistant P. aeruginosa strains was determined by broth microdilution. The presence of resistance genes was evaluated by PCR and DNA sequencing. Additionally, alterations in genes encoding P. aeruginosa outer membrane proteins were analyzed by PCR as well as SDS-PAGE. Clinical characteristics of the patients and the economic impact of hospitalization on the public health system were evaluated. PCR amplification showed that the blaKPC-2 and blaTEM genes were identified in three isolates (11%) and blaSHV gene in two isolates (7%). Outer membrane profiles obtained by SDS–PAGE indicated that the OprD porin was either absent or was produced at very low levels. A PCR assay using oprD-specific primers failed to show the presence of mutations in this gene. P. aeruginosa strains were isolated from 28 patients, among whom 43% (12/28) had sepsis, 31% (9/28) had respiratory failure, and 31% (9/28) had systemic arterial hypertension. A high mortality rate (39%) was observed in these patients, with an average duration of hospitalization of 34.6 days and a median cost of 3.275 dollars per patient. The production of carbapenemase was not the main mechanism of resistance in these strains. All carbapenem-resistant P. aeruginosa were isolated from patients hospitalized in the ICU. Besides the high mortality rate, many patients remained hospitalized for several days, resulting in a high cost of hospitalization for the public health system. Therefore, the evolution of this resistance and its dissemination should be actively monitored among critically ill patients to improve their health conditions

Identification of Sao Paulo metallo-beta-lactamase-1-producing Pseudomonas aeruginosa in the Central-West region of Brazil: a case study

Metallo-beta-lactamase production is an important mechanism for carbapenem resistance of Pseudomonas aeruginosa, which represents an emerging public health challenge. We report the case of a patient admitted to an intensive care unit, with sepsis caused by multidrug-resistant Sao Paulo Metallo-beta-lactamase-1-producing P. aeruginosa. This is the first case of infection by this pathogenic strain in the State of Mato Grosso do Sul, Brazil. Thus, infection control measures are required for preventing future spread and outbreaks.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundacao Univ Fed Grande Dourados, Lab Pesquisa Ciencias Saude, Dourados, MS, BrazilFundacao Univ Fed Grande Dourados, Hosp Univ Dourados, Dourados, MS, BrazilUniv Fed Sao Paulo, Dept Med, Lab Alerta, Disciplina Infectol, Sao Paulo, SP, BrazilLaboratório Alerta, Disciplina de Infectologia, Departamento de Medicina, Universidade Federal de São Paulo, São Paulo, SP, BrasilCNPq: 480949/2013-1FUNDECT: 05/2011FUNDECT: 04/2012CNPq: 07816/2009-5Web of Scienc

Detection of Treponema pallidum in whole blood samples of patients with syphilis by the polymerase chain reaction

Syphilis is caused by the bacterium Treponema pallidum. The diagnosis is based on clinical data and serological analysis; however, the sensitivity and specificity of such tests may vary depending on the type of test and stage of the infection. In order to overcome this premise, this study utilized the polymerase chain reaction (PCR) for the detection of T. pallidum DNA in whole blood samples of patients with syphilis. The blood samples from patients with or without symptoms of syphilis, but with positive results in enzyme-linked immunosorbent assay (ELISA), were included in this study. A venereal disease research laboratory (VDRL) test was performed for all collected sera samples. For PCR, the T. pallidum DNA was extracted from the collected blood samples and a specific primer set was designed to amplify 131 nucleotides of polA (Tp0105). The specificity of the primers was evaluated with the DNA of 17 different pathogens. From a total of 314 blood samples reactive in ELISA, 58.2% (183/314) of the samples were reactive in the VDRL test. In the PCR, 54% (168/314) of the ELISA-reactive samples were positive. In both tests (VDRL and PCR) 104 samples were positive. Of 104 positive samples for both tests, 71 were at the latent stage. Based on these results, it can be concluded that PCR with the designed set of primers can be utilized as a diagnostic method for T. pallidum detection in blood samples of patients with syphilis, especially those with latent infection. In addition, it can be utilized as a supplement for serological methods to improve the diagnosis of syphilis

The first year of the COVID-19 pandemic in an indigenous population in Brazil: an epidemiological study

This cross-sectional observational study that describes the epidemiological data of the first year of the COVID-19 pandemic in the Mato Grosso do Sul State, aimed to demonstrate the differences between indigenous and non-indigenous populations, characterize confirmed cases of COVID-19 according to risk factors related to ethnicity, comorbidities and their evolution and to verify the challenges in facing the disease in Brazil. SIVEP-Gripe and E-SUS-VE, a nationwide surveillance database in Brazil, from March 2020 to March 2021 in Mato Grosso do Sul state, were used to compare survivors and non-survivors from indigenous and non-indigenous populations and the epidemiological incidence curves of these populations. A total of 176,478, including 5,299 indigenous people, were confirmed. Among the indigenous population, 52.5% (confidence interval [CI] 51.2-53.9) were women, 38% (CI 36.7-39.4) were 20-39 years old, 56.7% were diagnosed by rapid antibody tests, 12.3% (CI 95%:11.5-13.2) had at least one comorbidity, and 5.3% (CI 95%:4.7–5.9) were hospitalized. In the non-indigenous patients, 56.8% were confirmed using RT-PCR, 4.4% (CI 95%:4.3-4.5) had at least one comorbidity, and 8.0% (CI 95%:7.9-8.2) were hospitalized. The majority of non-survivors were ≥60 years old (65.1% indigenous vs. 74.1% non-indigenous). The mortality in indigenous people was more than three times higher (11% vs. 2.9%). Indigenous people had a lower proportion of RT-PCR diagnoses; deaths were more frequent in younger patients and were less likely to be admitted to hospital. Mass vaccination may have controlled the incidence and mortality associated with COVID-19 in this population during the period of increased viral circulation