Neumonía en el paciente crítico: diagnóstico sindrómico rápido de PCR múltiple en un hospital general de agudos / Pneumonia in the critically ill patient: rapid syndromic diagnosis by multiplex PCR in a general hospital for acute care

Está demostrado en diversos estudios que los avances en el diagnóstico microbiológico reducen el tiempo de entrega de resultados y poseen un impacto clínico eviden-te. Hoy en día, las técnicas basadas en amplificación de ácidos nucleicos nos permiten hacer diagnóstico direc-tamente de la muestra y sumar la posibilidad de detectar más de un agente. Esto impacta tanto en el control de la multiresistencia (MR) como en el inicio de una terapéuti-ca apropiada. La implementación de un sistema de PCR múltiple rápido para neumonía puede ser útil en áreas crí-ticas, donde son frecuentes las infecciones respiratorias agudas (IRA) y el tiempo es un condicionante del éxito terapéutico. El objetivo de nuestro proyecto fue evaluar la implementación del diagnóstico sindrómico rápido por PCR múltiple para neumonía en el manejo del tratamiento de IRA en una unidad de cuidados intensivos. La con-ducta terapéutica fue la variable relevante. Este nuevo diagnóstico nos proporcionó una herramienta ágil, con un tiempo de respuesta de tres a cuatro horas. La ausencia o presencia de genes de resistencia y el microorganismo identificado fueron lo que condujo a la conducta terapéuti-ca acertada en el 75% de los casos. Constituyó una herra-mienta importante para el control de la multirresistencia bacteriana y aumentó la oportunidad de éxito terapéutico

It has been shown in various studies that advances in microbiological diagnosis reduce the delivery time of results and have an evident clinical impact. Today, techniques based on nucleic acid amplification allow us to diagnose directly from the sample and add the possibility of detecting more than one agent. This impacts both the control of MR and the initiation of appropriate therapy. The implementation of a rapid multiplex PCR system for pneumonia can be useful in critical areas where acute respiratory infections (ARI) are frequent and time is a determining factor for therapeutic success. The objective of our project was to evaluate the implementation of rapid syndromic diagnosis by multiple PCR for pneumonia in the management of ARI treatment in an Intensive Care Unit. The therapeutic behavior was the relevant variable. This new diagnosis provided us with an agile tool, with a response time of 3 to 4 hours. The absence or presence of resistance genes and the identified microorganism was what led to the correct therapeutic approach in 75% of the cases. It constituted an important tool for the control of bacterial multiresistance and increased the opportunity for therapeutic success.

Development of a multiplex PCR assay for the detection of metallo-beta-lactamase genes in Pseudomonas aeruginosa / Desarrollo de un PCR múltiple para la detección de genes metalo-beta-lactamasa en Pseudomonas aeruginosa

SUMMARY: The appearance of Pseudomonas aeruginosa strains with multi-resistance to antibiotics is a clinical problem of great relevance. The methods for detecting these resistances are laborious and slow, which is a complication when treating patients promptly. In this work, we developed a simple method for simultaneous detection of several carbapenem resistance genes using a multiplex PCR assay. The PCR assay developed, followed by electrophoretic separation of fragments, allows to simultaneously identify the presence of 6 antibiotic resistance genes: bla-VIM (261 bp), bla-IMP (587 bp), bla-SPM (648 bp), bla-GIM-1 (753 bp), bla-NDM-1 (813 bp) and bla-KPC (882 bp). We analyzed 7 clinical isolates of P. aeruginosa obtained in Chile, finding the resistance genes bla-VIM, bla-IMP, bla-SPM, bla-GIM, and bla-NDM in 5 of them. We found a perfect correlation between the detection of various resistance genes by PCR and the results obtained by antibiograms. Interestingly, 2 of the strains possessed 3 different resistance genes simultaneously. Finally, in this work, we found the presence of 3 genes never described before in clinical isolates of P. aeruginosa in Chile (bla-IMP, bla-SPM, and bla-GIM-1). We developed a rapid multiplex PCR test for the simultaneous detection of up to 6 antibiotic resistance genes of the metallo-β-lactamase family in P. aeruginosa.

La aparición de cepas de Pseudomonas aeruginosa con resistencias a diversos antibióticos es un problema clínico de gran relevancia. Los métodos de detección de dichas resistencias son laboriosos y lentos, lo que genera una complicación al momento de tratar a los pacientes oportunamente. En este trabajo desarrollamos un método simple de detección simultánea de varios genes de resistencia a carbapenem, mediante un sistema de PCR múltiple. El ensayo de PCR desarrollado, seguido de una separación electroforética de los amplicones, permite distinguir simultáneamente la presencia de 6 genes de resistencia a antibióticos: bla-VIM (261 pb), bla-IMP (587 pb), bla-SPM (648 pb), bla-GIM-1 (753 pb), bla-NDM-1 (813 pb) y bla-KPC (882 pb). Analizamos 7 aislados clínicos obtenidos en Chile, encontrando en 5 de ellos los genes de resistencia bla-VIM, bla-IMP, bla-SPM, bla-GIM y bla-NDM. Encontramos una perfecta correlación entre la detección de diversos genes de resistencia y los resultados obtenidos mediante antibiogramas. Interesantemente, 2 de las cepas mostraron poseer simultáneamente 3 genes de resistencia distintos. Por último, en este trabajo encontramos la presencia de 3 genes nunca antes descritos en aislados clínicos de P. aeruginosa en Chile (bla-IMP, bla-SPM y bla-GIM-1). Hemos desarrollado un test rápido de PCR múltiple, para la detección simultánea de hasta 6 genes de resistencia a antibióticos de la familia.a de las metallo-b-lactamases en P. aeruginosa.

Establishment of a PCR-SSP method for the simultaneous amplification and identification of the presence of KIR genes / 中华医学遗传学杂志

OBJECTIVE@#To develop a polymerase chain reaction-sequence specific primer (PCR-SSP) method for simultaneous amplification and identification of the KIR genes among Chinese population.@*METHODS@#Peripheral blood samples from 132 healthy donors who had given blood at Shenzhen Blood Center from January 2015 to November 2015 were selected as the study subjects. Based on the polymorphism and single nucleotide polymorphism (SNP) information of high-resolution KIR alleles in the Chinese population and the IPD-KIR database, specific primers were designed to amplify all the 16 KIR genes and the 2DS4-Normal and 2DS4-Deleted subtypes. The specificity of each pair of PCR primers was verified by using samples with known KIR genotypes. During PCR amplification of the KIR gene, co-amplification the fragment of human growth hormone (HGH) gene by multiplex PCR was used as the internal control to prevent false negative results. A total of 132 samples with known KIR genotypes were randomly selected for blind inspection to verify the reliability of the developed method.@*RESULTS@#The designed primers can specifically amplify the corresponding KIR genes, with clear and bright bands for the internal control and KIR genes. The results of detection are fully consistent with the known results.@*CONCLUSION@#The KIR PCR-SSP method established in this study can yield accurate results for the identification of the presence of KIR genes.

Application of multiplex PCR capillary electrophoresis and analysis of distribution characteristics of HPV infection subtypes / 中华预防医学杂志

Objective: To analyze the characteristics of human papillomavirus (HPV) infection and the distribution of HPV subtypes in Shijiazhuang, Hebei Province, and to explore the application evaluation of multiple PCR capillary electrophoresis fragment analysis for HPV typing test. Methods: A population-based cross-sectional study was conducted among 434 women (age range 17 to 74 years old, 260 patients and 174 physical examinations) included from May to August 2022 in Hebei General Hospital. HPV typing was detected by multiple PCR-capillary electrophoresis fragment analysis. Using the multiple fluorescence quantitative PCR kit as a reference, Chi-square test was used to analyze the diagnostic effect of multiple PCR-capillary electrophoresis fragment analysis, and the consistency was analyzed by Kappa value. Results: The total HPV infection rate was 45.85%(199/434), including 35.48% (154/434) of high-risk HPV (HR-HPV), 3.92% (17/434) of low-risk HPV (LR-HPV), 6.45% (28/434) of HR-HPV and LR-HPV mixed infection, 27.88% (121/434) of single type HPV and 17.97% (78/434) of multi type HPV. HPV52 (9.68%, 42/434), HPV16 (6.91%, 30/434), and HPV58 (6.91%, 30/434) are common HPV subtypes. The positive rate of physical examination was 45.40% (79/174), which was slightly lower than that of patients 46.15% (120/260), there was no significant difference (χ2=0.024,P>0.05). The highest infection rate in the 17-30 age group was 54.76% (46/84), and there was no statistical difference among the age groups(χ2=4.123,P>0.05). The sensitivity and specificity of multiplex PCR capillary electrophoresis fragment analysis were 92.96% and 94.04%, respectively, and Kappa value was 0.870, with the multiplex fluorescent quantitative PCR as the reference. Conclusion: HPV infection may appear younger, and the positive rate of HR-HPV infection is the highest, with HPV52, 16, 58 as the main infection subtypes. The detection results of multiplex PCR capillary electrophoresis fragment analysis method are highly consistent with those of multiplex fluorescent quantitative PCR method, which is suitable for HPV DNA typing.

Padronização de uma PCR multiplex para a detecção de fraude em leite bubalino e caprino por adição de leite bovino / Standardization of a multiplex PCR for detection of fraud in buffalo and goat milk by addition of bovine milk

O consumo de leite de espécies como bubalino e caprino tem se popularizado por representarem uma alternativa para indivíduos que possuem restrições alimentares relacionadas ao leite bovino e em virtude das propriedades nutricionais desses alimentos. No entanto, fatores como a baixa produção e a sazonalidade predispõem a adulterações destes alimentos, principalmente pela adição de leite bovino, visando maior rendimento e lucratividade. Assim, o objetivo do estudo foi padronizar um método de PCR multiplex para autenticação de leites bubalino e caprino. Para isso, amostras de leite exclusivamente de cada espécie foram utilizados para a padronização da técnica. Em seguida, foi realizada a fraude pela adição de leite bovino ao caprino e ao bubalino, em proporções de 0,1% até 100%. A técnica foi eficaz, precisa, rápida e prática para a detecção do DNA de bovino, bubalino e caprino, separadamente e em conjunto. Na fraude experimental, o limite de detecção da técnica ocorreu a partir do menor percentual testado (0,1%) tanto no leite caprino quanto no bubalino. Dessa forma, a PCR multiplex testada mostrou ser uma importante ferramenta para a autenticação de leite, pendendo ser utilizada para fins de fiscalização por órgãos competentes.

Milk consumption of species such as buffalo and goat has become popular due to the nutritional properties of these foods and because they represent an alternative for individuals who have dietary restrictions related to bovine milk. However, factors such as low production and seasonality predispose to adulteration, mainly by the addition of bovine milk, aiming at higher yield and profitability. Thus, the aim of the present study was to standard a multiplex PCR method for buffalo and goat milks authentication. For this, the milks exclusively of each species were used to standardize the technique. Subsequently, fraud was performed by the addition of bovine milk to goat and buffalo in proportions from 0.1% to 100%. The technique was effective and accurate for detecting bovine, buffalo and goat DNA separately and together quickly and practically. In experimental fraud, the detection limit of the technique occurred from the lowest percentage tested (0.1%) in both goat and buffalo milk. Thus, the multiplex PCR tested proved to be an important tool for milk authentication, pending to be used for supervision by competent agencies.

Utilidad del panel de PCR multiplex en el diagnóstico microbiológico temprano y adecuación antimicrobiana en pacientes críticos con neumonía / Usefulness of the multiplex PCR panel in the early microbiological diagnosis and antimicrobial suitability in critical patients with pneumonia / Utilidade do painel de PCR multiplex no diagnóstico microbiológico precoce e adequação antimicrobiana em pacientes críticos com pneumonia

Resumen: Introducción: el inicio temprano de la antibioticoterapia adecuada en infecciones graves se asocia con reducción de la mortalidad. La identificación precoz del microorganismo es fundamental para realizar un tratamiento dirigido y disminuir la terapéutica inicial inapropiada. Objetivo: valorar la utilidad de una técnica de biología molecular por amplificación de ácidos nucleicos mediante reacción en cadena de polimerasa en tiempo real para diagnóstico microbiológico temprano y adecuación de la antibioticoterapia en pacientes con neumonías graves. Metodología: estudio retrospectivo observacional llevado a cabo en la unidad de cuidados intensivos del Hospital Maciel. Se analizaron muestras respiratorias de pacientes con diagnóstico o sospecha de neumonía. Se compararon los resultados microbiológicos obtenidos por técnicas convencionales y por biología molecular multiplex (panel neumonía). Resultados: se incluyeron 53 muestras obtenidas de 51 pacientes. El multiplex detectó al menos un microorganismo en 38 (71,7%) muestras frente a 30 (56.6%) desarrollos en cultivos tradicionales. La mayoría de las muestras se obtuvieron bajo antibioticoterapia previa (86.8%). El panel neumonía mostró un porcentaje de concordancia positiva combinado de 100% y un porcentaje de concordancia negativa del 94% para la identificación bacteriana en comparación con los métodos microbiológicos tradicionales. En 27 (51%) casos el resultado del panel de neumonía determinó un cambio en la conducta terapéutica. Conclusiones: la técnica de PCR permite la identificación temprana de microorganismos causantes de neumonía optimizando la terapéutica empírica inicial y racionalizando el uso de antimicrobianos. Un panel negativo aleja el planteo de infección respiratoria a gérmenes habituales y permite considerar diagnósticos diferenciales en cuanto a foco y/o etiología.

Summary: Introduction: the early initiation of the adequate antibiotic therapy in severe infections is associated to a reduction in mortality. Early identification of the microorganism is essential to define directed therapy and decrease the initial inadequate treatment. Objective: to assess usefulness of a molecular biology technique by nucleic acid amplification through a polymerase chain reaction in real time for an early microbiological diagnosis and correction of the antibiotic therapy in patients with severe pneumonias. Method: retrospective, observational study conducted in the intensive care unit of Maciel Hospital. The respiratory samples of patients with a diagnosis of pneumonia or suspicious to have pneumonia were analyzed. The microbiological results obtained were compared using conventional techniques and multiplex molecular biology (pneumonia panel). Results: 53 samples obtained from 51 patients were included in the study. Multiplex detected at least one microorganism in 38 (71.7%) samples compared to 30 (56.6%) in traditional cultures. Most samples were obtained under the previous antibiotic therapy (86.8%). The pneumonia panel showed a combined positive agreement percentage of 100% and a negative agreement of 94% for the identification of bacteria when compared to the traditional microbiological methods. In 27 cases (51%) the pneumonia panel results determined changing the therapeutic behavior. Conclusions: the PCR technique allows for the early identification of microorganisms causing pneumonia, thus optimizing initial empirical therapy and rationalizing the use of antibiotics. A negative panel reduces the suspicion of a respiratory infection caused by the usual germs and enables considering differential diagnosis in terms of etiology or cause.

Resumo: Introdução: o início precoce da antibioticoterapia adequada em infecções graves está associado à redução da mortalidade. A identificação precoce do microrganismo é essencial para realizar o tratamento dirigido e reduzir o uso inicial inadequado de antimicrobianos. Objetivo: avaliar a utilidade de uma técnica de biologia molecular para amplificação de ácidos nucleicos por reação em cadeia da polimerase em tempo real para diagnóstico microbiológico precoce e adequação da antibioticoterapia em pacientes com pneumonia grave. Metodologia: estudo observacional retrospectivo realizado na unidade de terapia intensiva do Hospital Maciel. Amostras respiratórias de pacientes com diagnóstico ou suspeita de pneumonia foram analisadas. Os resultados microbiológicos obtidos por técnicas convencionais e por biologia molecular multiplex (painel de pneumonia) foram comparados. Resultados: foram incluídas 53 amostras obtidas de 51 pacientes. O multiplex detectou pelo menos um microrganismo em 38 (71,7%) amostras em comparação com 30 (56,6%) usando culturas tradicionais. A maioria das amostras foi obtida com antibioticoterapia prévia (86,8%). O painel de pneumonia mostrou uma concordância percentual positiva combinada de 100% e uma concordância percentual negativa de 94% para identificação bacteriana em comparação com métodos microbiológicos tradicionais. Em 27 (51%) casos, o resultado do painel de pneumonia determinou mudança no comportamento terapêutico. Conclusões: a técnica de PCR permite a identificação precoce de microrganismos causadores de pneumonia, otimizando a terapia empírica inicial e racionalizando o uso de antimicrobianos. Um painel negativo afasta a suspeita de infecção respiratória pelos germes usuais e permite considerar diagnósticos diferenciais em termos de foco e/ou etiologia.

Investigação Citogenômica de Crianças com Doença Cardíaca Congênita: Experiência de um Centro no Brasil / Cytogenomics Investigation of Infants with Congenital Heart Disease: Experience of a Brazilian Center

Resumo Fundamento Algumas síndromes têm características específicas e facilmente reconhecíveis, enquanto outras podem ser mais complexas de se identificar e podem apresentar diferentes manifestações fenotípicas, por exemplo. Um diagnóstico etiológico é importante para entender a natureza da doença, para estabelecer o prognóstico e para começar o tratamento, permitindo a inclusão de pacientes na sociedade e reduzindo o custo financeiro dessas doenças. Objetivo A proposta inicial deste estudo foi a triagem citogenética para detectar a síndrome de deleção 22q11.2 (SD22q11.2) em recém-nascidos e crianças com doença cardíaca congênita utilizando a técnica da amplificação multiplex de sondas dependente de ligação (MLPA). Assim, por meio da pesquisa, outras mudanças genômicas foram identificadas nesses pacientes cardíacos. Nosso objetivo se estendeu a investigar essas outras mudanças citogenéticas. Métodos Investigamos 118 recém-nascidos com doenças cardíacas congênitas nascidos consecutivamente durante um ano, utilizando a técnica da MLPA. Resultados A técnica da MLPA permitiu a detecção da SD22q11.2 em 10/118 pacientes (8,5%). Outras alterações genômicas foram identificadas em 6/118 pacientes (5%): 1p36 del, 8p23 del (2 casos), 7q dup, 12 dup e 8q24 dup. Conclusão Este estudo ressalta a relevância da detecção de alterações genômicas que estão presentes em recém-nascidos e crianças com doenças cardíacas congênitas por meio de ferramentas citogenômicas.

Abstract Background Some syndromes have specific and easily recognizable features, while others may be more complex to identify and may present different phenotypic manifestations, for example. An etiological diagnosis is important to understand the nature of the disease, to establish the prognosis and to start the treatment, allowing the inclusion of patients in society and reducing the financial cost of such diseases. Objective The initial proposal of this study was cytogenetic screening for the detection of the 22q11.2 deletion syndrome in consecutive newborns and infants with congenital heart disease using the multiplex ligation-dependent probe amplification (MLPA) technique. Therefore, throughout our research, other genomic alterations were identified in these cardiac patients. Thus, our objective was extended to investigate these other cytogenetic alterations. Methods We investigated 118 neonates with congenital heart diseases born consecutively during one year using the MLPA technique. Results The MLPA technique allowed the detection of 22q11.2DS in 10/118 patients (8.5%). Other genomic alterations were also identified in 6/118 patients (5%): 1p36 del, 8p23 del (2 cases), 7q dup, 12 dup and 8q24 dup. Conclusion This study highlights the relevance of detecting genomic alterations that are present in newborns and infants with congenital cardiac diseases using cytogenomic tools.

Comparison of Application of MD-VF-Auto SEM Method and Plankton Gene Multiplex PCR System in the Diagnosis of Drowning / 法医学杂志

OBJECTIVES@#To compare the application effect of microwave digestion - vacuum filtration - automated scanning electron microscopy (MD-VF-Auto SEM) method and plankton gene multiplex PCR system in the diagnosis of drowning.@*METHODS@#Lung, liver and kidney tissue of 10 non-drowning cases and 50 drowning cases were prepared for further MD-VF-Auto SEM method analysis and plankton gene multiplex PCR system analysis. The positive detection rate of the two methods in each tissue was calculated.@*RESULTS@#The positive rate of the MD-VF-Auto SEM method detecting diatoms in drowning cases was 100%, and few diatoms were detected in the liver and kidney tissues of 6 non-drowning cases. By using the plankton gene multiplex PCR system, the diatom positive rate of drowning cases was 84%, and all the non-drowning cases were negative. There were significant differences in the positive rate of the liver, kidney tissues between MD-VF-Auto SEM method and plankton gene multiplex PCR system (P<0.05), as well as the total positive rate of cases. However, no significant differences were found in the positive rates of lung tissues (P>0.05).@*CONCLUSIONS@#MD-VF-Auto SEM method is more sensitive than plankton gene multiplex PCR system in diatom test. But the plankton gene multiplex PCR system can also detect plankton other than diatoms. Combination of the two methods can provide a more reliable basis for the diagnosis of drowning.

Caracterização molecular e fenotípica de Klebsiella spp. produtoras de NDM / Molecular and phenotypic characterization of Klebsiella spp. NDM producers

O perfil de resistência, que algumas das espécies do complexo Klebsiella pneumoniae podem expressar, representa uma grande ameaça à saúde humana, particularmente quando resistentes aos carbapenêmicos, que são amplamente utilizados no tratamento de infecções graves em pacientes hospitalizados. O principal mecanismo de resistência aos carbapenêmicos é a produção de carbapenemases, particularmente dos tipos KPC e NDM. Um dos compostos desenvolvidos para o tratamento de infecções causadas por cepas produtoras de KPC é a combinação ceftazidimaavibactam (CAZ-AVI), mas que não tem atividade inibitória sobre metalo-betalactamases, a exemplo das NDMs. Os objetivos deste trabalho foram determinar a frequência das espécies do complexo K. pneumoniae e da coprodução de KPC, avaliar a clonalidade dos isolados, a sensibilidade ao aztreonam-avibactam (ATM-AVI), o desempenho do disco de meropenem (MEM) com inibidores para detecção de coprodução de NDM e KPC e desenvolver um teste de triagem para prever a sensibilidade ao ATM-AVI. Um total de 113 isolados do complexo K. pneumoniae produtoras de NDM ou coprodutoras de NDM e KPC, provenientes da coleção de bactérias do Grupo Fleury, coletadas períodos pré e pós início do uso de CAZ-AVI no Brasil, foram utilizadas neste estudo. A identificação da espécie e a presença dos genes blaNDM e blaKPC foi confirmada por PCR multiplex. A clonalidade dos isolados foi avaliada por eletroforese em campos pulsados (PFGE) após clivagem com XbaI. A produção de carbapenemases foi confirmada utilizando-se o teste Blue Carba. O desempenho dos discos de meropenem e CAZ-AVI contendo um ou mais inibidores de carbapenemases foi comparado com o teste molecular. A pré-difusão combinada foi realizada pré-incubando-se o ágar não inoculado com disco de CAZ-AVI, e a seguir aplicando-se o inóculo bacteriano e um disco de ATM após remover o disco de CAZ-AVI. Após incubação, os halos foram aferidos e correlacionados com a concentração inibitória mínima para ATM-AVI. As CIMs para ATM e ATM-AVI foram determinadas segundo o EUCAST. A identificação das espécies por PCR evidenciou as seguintes frequências: K. pneumoniae 75,2% (n=85); K. quasipneumoniae 16,8% (n=19), e K. variicola 8% (n=9). Uma fração de 12,4% (n=14) dos isolados apresentaram os genes blaNDM e blaKPC e 87,6% (n=99) apenas blaNDM. A análise dos perfis de PFGE de K. pneumoniae evidenciou a presença de cinco grupos clonais predominantes. Isolados do principal grupo clonal Ap (n=15) foram detectados nas cidades de São Paulo e Porto Alegre durante todo o período analisado. O grupo clonal Lp foi detectado nas cidades de São Paulo e Recife em 2019. Os dois principais grupos clonais no período pré-CAZ-AVI continham maior número de isolados do que aqueles no período de uso do CAZ-AVI. Os perfis de PFGE de K. quasipneumoniae evidenciaram quatro grupos clonais predominantes, e presentes apenas no estado de São Paulo, com persistência do grupo clonal Aq desde 2017. Quanto à K. variicola, foram observados dois grupos clonais predominantes Av e Bv, o primeiro presente apenas em São Paulo desde 2018 e o segundo em Porto Alegre apenas em 2019. Calculando-se a diferença entre os diâmetros de halo do disco MEM contendo EDTA e ácido fenilborônico (AFB) e o maior dos halos obtidos para MEM com EDTA ou AFB, observou-se que todos os isolados com coexpressão de KPC e NDM apresentaram diferença ≥ 5 mm. Uma fração de 42,3% dos isolados positivos apenas para blaNDM apresentaram sensibilidade para ATM (CIM ≤ 4 mg/L). Todos os isolados testados apresentaram CIM para ATM-AVI ≤ 1/4 mg/L, sendo a CIM90 0,125/4 mg/l. No teste de pré-difusão combinada, o menor diâmetro de halo obtido foi de 23 mm. A espécie predominante na amostragem foi K. pneumoniae. A disseminação clonal, observada neste estudo, contrasta com a diversidade clonal descrita em outros locais do mundo para produtores de NDM, exceto Grécia e China. Considerando os pontos de corte atuais para ATM, é provável que haja resposta clínica adequada no uso de ATM-AVI no tratamento de infecções causadas por isolados produtores de NDM e coprodutores de KPC e NDM. Utilizando-se o valor de corte de ≤ 5 mm para a diferença entre halos de inibição, de MEM com AFB e EDTA e o segundo maior halo com inibidor, a sensibilidade foi de 100% e a especificidade foi de 96,1,0%. O método de pré-difusão com CAZ-AVI e ATM é um método simples e o diâmetro ≥ 23 mm tem excelente correlação com a CIM para ATM-AVI ≤ 1/4 mg/L

The resistance profile, which some species of the Klebsiella pneumoniae complex may express, represent a great threat to human health, particularly when resistant to carbapenems, which are widely used in the treatment of severe infections in hospitalized patients. The main mechanism of resistance to carbapenems is the production of carbapenemases, particularly KPCs and NDMs. One of the compounds developed for the treatment of infections caused by KPC-producing strains is the combination ceftazidime-avibactam (CAZ-AVI), but which has no inhibitory activity on metallobetalactamases, as is the case for NDMs. The objectives of this work were to determine the frequency of K. pneumoniae complex species and KPC co-production, evaluate the clonality of isolates, the susceptibility to aztreonam-avibactam (ATM-AVI), the performance of meropenem (MEM) disks with inhibitors for detecting NDM co-production and KPC and develop a screening test to predict sensitivity to ATM-AVI. A total of 113 NDM-producing or NDM and KPC co-producing K. pneumoniae complexes, from the Fleury Group's bacteria collection, collected in the pre- and post-starting periods of CAZ-AVI use in Brazil, were used in this study. Species identification and the presence of the blaNDM and blaKPC genes were confirmed by multiplex PCR. The clonality of the isolates was evaluated by pulsed field electrophoresis (PFGE) after cleavage with XbaI. Carbapenemase production was confirmed using the Blue Carba test. The performance of MEM and CAZ-AVI disks containing one or more carbapenemase inhibitors was compared with the molecular test. Combined pre-diffusion was performed by preincubating the uninoculated agar with a CAZ-AVI disk, and then applying the bacterial inoculum and na ATM disk after removal of the CAZ-AVI disk. After incubation, halos were measured and correlated with the minimum inhibitory concentration (MIC) for ATM-AVI. ATM and ATM-AVI MICs were determined according to EUCAST. The identification of species by PCR evidenced the following frequencies: K. pneumoniae 75.2% (n=85); K. quasipneumoniae 16.8% (n=19), and K. variicola 8% (n=9). A fraction of 12.4% (n=14) of the isolates had the blaNDM and blaKPC genes and 87.6% (n=99) had only blaNDM. The analysis of the PFGE profiles of K. pneumoniae evidenced the presence of five predominant clonal groups. Isolates from the main clonal group Ap (n=16) were detected in the cities of São Paulo and Porto Alegre throughout the analyzed period. The clonal group Lp was detected in the cities of São Paulo and Recife 2019. The PFGE profiles of K. quasipneumoniae showed four predominant clonal groups, present only in the state of São Paulo, with persistence of the clonal group Aq since 2017. As for K. variicola, two predominant clonal groups Av and Bv were observed, the first present only in São Paulo since 2018 and the second in Porto Alegre only in 2019. Calculating the difference between the inhibition zone diameters of the MEM disk containing EDTA and phenylboronic acid (AFB) and the largest of the inhibition zone diameters obtained for MEM with EDTA or AFB, it was observed that all isolates with co-expression of KPC and NDM showed a difference 5 ≥mm. A fraction of 42.3% of isolates positive only for blaNDM showed sensitivity to ATM (MIC ≤ 4 mg/L). All tested isolates presented MIC for ATM-AVI ≤ 1/4 mg/L, being the MIC90 0.125/4 mg/l. In the combined pre-diffusion test, the smallest inhibition zone diameter obtained was 23 mm. The predominant species in the sample was K. pneumoniae, but a significant fraction of the other species in the complex was also observed in the sample. The clonal spread observed in this study contrasts with the clonal diversity described elsewhere in the world for NDM-producing isolates, except Greece and China. Considering the current cut-off points for ATM, it is likely that there is an adequate clinical response in the use of ATM-AVI in infections caused by NDM-producing and KPC-NDM co-producing isolates in Brazil. Using the cutoff value of 5 mm for the difference between inhibition zones, of MEM with AFB and EDTA and the second largest zone of MEM with inhibitor, the sensitivity was 100% and the specificity was 96.1%. The pre-diffusion method with CAZ-AVI and ATM is a simple method and the diameter ≥ 23 mm has excellent correlation with the MIC for ATM-AVI ≤ 1/4 mg/L

Étiologies des méningites et méningoencéphalites dans les services de réanimation polyvalente à Libreville de 2020 à 2021 / Etiologies of meningitis and meningoencephalitis in intensive care units of Libreville from 2020 to 2021

Introduction. Les méningites/méningo-encéphalites sont des urgences médicales d'étiologies variées. La technique de diagnostic Multiplex Polymerase Chain Reaction (PCR) permet de détecter la présence de bactéries et de virus dans le liquide céphalorachidien (LCR) avec une spécificité et une sensibilité ≥ 90%. L'objectif de cette étude était d'identifier en utilisant cette technique, les principaux germes responsables des méningites et méningo-encéphalites en réanimation à Libreville. Patients et méthodes. Nous avons mené une étude transversale allant d'octobre 2020 à septembre 2021. Les critères d'inclusion étaient : être admis en réanimation au CHUL et à l'HIAOBO pour suspicion de méningite ou méningo-encéphalite, obtenir l'accord des familles pour l'analyse du liquide céphalorachidien (LCR) par multiplex PCR. Les variables étudiées étaient : la fréquence, les données sociodémographiques, les aspects cliniques et paracliniques. Résultats. Soixante et onze patients ont répondu aux critères d'inclusion. L'âge moyen était de 21,1 ± 10,4 ans et le sex ratio de 1,2. Les motifs d'admission étaient l'altération de l'état de conscience (77%) et l'état de mal épileptique (21%). Plasmodium falciparum a été retrouvé seul chez 38 patients (53,5%) et associé à Listeria monocytogenes chez 4 patients (1,4%). Les méningo-encéphalites à Herpès simplex virus ont été observées chez 4 patients (1,4%) dont l'âge variait entre 40 ans et moins de 50 ans. Un patient (1,4%) présentait une coinfection à S. épidermidis, flavivirus et alphavirus. Des méningo-encéphalites sans germes ont été observées chez 5 patients (%). Conclusion. Le principal germe responsable de méningoencéphalite en réanimation à Libreville est P. falciparum. Des virus tels que le flavivirus et l'alphavirus non détectés par les méthodes usuelles ont aussi été mis en évidence grâce au multiplex PCR.

Introduction. Meningitis/meningoencephalitis are medical emergencies of various etiologies. The Multiplex Polymerase Chain Reaction (PCR) technique allows the detection of the presence of bacteria and viruses in the cerebrospinal fluid (CSF) with a specificity and sensibility of above 90%. The aim of this study was to identify the most common germs responsible for meningitis and meningoencephalitis in the intensive care units of Libreville using this technique,. Patients and methods. We conducted a transversal study from October 2020 to September 2021. Inclusion criteria were: being admitted to intensive care unit of CHUL and HIAOBO for suspicion of meningitis or meningoencephalitis and having the parent's approval for multiplex PCR analysis of CSF. Variables studied included frequency, sociodemographic data, clinical and paraclinical aspects. Results. Seventy one patients were included. Mean age was 21.1 ± 10.4 years and the sex ratio was 1.2. Reasons for admission were altered consciousness (77%) and epilepsy (21%). Plasmodium (P) faciparum was detected alone in 38 cases (53.5%) and associated to Listeria monocytogenes in 4 patients (5.6%). Herpex simplex viral meningoencephalitis was observed in 4 patients (5.6%) aged between 40 and less than 50 years. One patient (1.4%) had co-infection with S. epidermidis, flavivirus and alphavirus. Meningoencephalitis with no germs was found in 5 patients (7%). Conclusion. The main etiology of meningoencephalitis in intensive care units of Libreville is P. falciparum. Viruses not detected by usual methods like flavivirus and alphavirus were detected by multiplex PCR.

Identificación molecular y diversidad genética de las especies de nematodos Globodera rostochiensis y G. pallida en regiones productoras de papa de Guatemala / Molecular identification and genetic diversity of the species of nematodesGlobodera rostochiensisandG. pallidain potato growing regions of Guatemala

En Guatemala, la producción del cultivo de papa se ve afectada por los nematodos Globodera rostochiensis y Globo-dera pallida. La capacidad de ambas especies para formar quistes complica su control y provoca el aumento de sus poblaciones. En Guatemala se reporta la presencia de ambas especies de nematodos por identificación morfológica, sin embargo, no se ha realizado una confirmación molecular. Este es el primer estudio para validar la presencia de ambas especies de nematodos por PCR múltiple y la determinación de la diversidad y estructura genética de las poblaciones utilizando marcadores moleculares. Se realizaron muestreos en cuatro departamentos productores de papa del país. La identificación por PCR se realizó con el cebador común ITS5 y los cebadores PITSr3 específico para G. rostochiensisy PITSp4 para G. pallida. La caracterización molecular se realizó con el marcador AFLP. Se confirmó la presencia de las dos especies de nematodos en los cuatro departamentos. Los índices de diversidad Shannon y heterocigosidad esperada revelaron mayor diversidad genética en G. rostochiensis (H = 0.311, He = 0.301) que en G. pallida (H = 0.035, He = 0.223). Los métodos NJ, DAPC y PCA exhibieron una débil estructura entre las poblaciones de ambas especies de nematodos. Los resultados sugieren un patrón de dispersión desde Quetzaltenango hacia el resto del país, atribuido a la comercialización de semilla contaminada con nematodos. Se sugiere promover programas de socialización sobre los beneficios del uso de semilla certificada, además de constantes monitoreos moleculares para un diagnóstico certero de ambas especies de nematodos.

In Guatemala, potato crop production is affected by the nematodes Globodera rostochiensis and Globodera pallida. The ability of both species to form cysts complicates their control and causes an increase in their populations. In Guatemala, both species of nematodes have been reported by morphological identification; however, molecular confirmation has not been carried out. It is the first study to validate the presence of both nematode species by multiplex PCR and determine the diversity and genetic structure of the populations using molecular markers. Sampling was carried out in four pota-to-producing departments of the country. PCR identification was performed with the common primer ITS5 and the primers PITSr3 specific for G. rostochiensis and PITSp4 for G. pallida. We performed molecular characterization with the AFLP marker. We confirmed the presence of the two nematode species in the four departments. Shannon diversity and expected heterozygosity indices revealed higher genetic diversity in G. rostochiensis (H = 0.311, He = 0.301) than in G. pallida (H = 0.035, He = 0.223). The NJ, DAPC, and PCA methods exhibited weak structure among populations of both nematode species. The results suggest a dispersal pattern from Quetzaltenango to the rest of the country, attributed to the commer-cialization of seed contaminated with nematodes. We suggest promoting socialization programs on the benefits of using certified seeds and constant molecular monitoring for an accurate diagnosis of both species of nematodes.

Comparing multiplex and multiplex real-time polymerase chain reaction with traditional blood culture in bacterial detection among patients with septicemia

Aims@#This study was aimed to test the specificity of primers and probes with target genes by using multiplex PCR and multiplex real-time PCR methods. These methods were compared with traditional blood culture methods in detecting five bacteria causing sepsis, including Acinetorbacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus.@*Methodology and results@#A total of 587 blood samples from patients diagnosed with sepsis and septic shock were collected at Thanh Nhan Hospital, Hanoi, Vietnam. Each sample was divided into three parts for bacterial culture, multiplex PCR and multiplex real-time PCR to detect the similarity of the two PCR methods with the bacterial culture method. Conditions in multiplex PCR and multiplex real-time PCR were optimized to ensure the successful amplification of target genes. Results showed that the primers and probes were tested completely specific to the target genes and using multiplex PCR and multiplex real-time PCR techniques could detect five pathogens causing sepsis, including A. baumannii, K. pneumoniae, P. aeruginosa, E. coli and S. aureus.@*Conclusion, significance and impact of study@#Both multiplex PCR and multiplex real-time PCR methods have high similarities with the culture method, showing potential in the application of bacteria detection in sepsis.

Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp, and Listeria monocytogenes DNA in milk / [Padronização de um método rápido de PCR quadriplex para a detecção simultânea de DNA de bovino, bubalino, Salmonella spp. e Listeria monocytogenes em leite]

The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.

O objetivo do presente estudo foi padronizar um protocolo de reação em cadeia da polimerase (PCR) para a autenticação de leite bovino e bubalino e a detecção da presença de Salmonella spp. e Listeria monocytogenes. Para isso, o DNA-alvo foi extraído, misturado e submetido ao ensaio de PCR. Amostras de leite foram fraudadas e contaminadas experimentalmente com os micro-organismos, para se avaliar a detecção do DNA-alvo em diferentes tempos de cultivo, os títulos bacterianos e a concentração de material genético. Além disso, o protocolo foi testado com DNA extraído diretamente do alimento, sem a etapa de pré-enriquecimento. A PCR quadriplex proposta mostrou boa precisão na identificação de sequências de DNA-alvo. Foi possível identificar simultaneamente todas as sequências de DNA no momento da inoculação (0h), quando as amostras estavam contaminadas com 2 UFC/250mL, e com seis horas de cultura, quando o inóculo inicial foi de 1 UFC/250mL. Também foi possível detectar diretamente as sequências de DNA do alimento quando este foi inoculado com 3 UFC/mL de bactérias. Dessa forma, a metodologia proposta apresentou desempenho satisfatório, otimização do tempo de análise e potencial para detecção de micro-organismos em baixos títulos, podendo ser utilizada para detecção de fraude e contaminação.

ß-l; -lactamasas AmpC en bacilos gramnegativos de aislados clínicos en un centro hospitalario de tercer nivel en Colombia / AmpCß-l;-lactamases in Gram-negative bacilli from clinical isolates in a Colombian tertiary hospital

Introdución: Las ß-lactamasas AmpC son enzimas con capacidad hidrolítica, pueden ser de tipo constitutivo o inducible. No existe un método estandarizado para su determinación fenotípica por normas internacionales; la detección de estas mediante el uso de la biología molecular podría ser una alternativa útil para vigilancia y control de la diseminación de clones circulantes en el entorno hospitalario. Objetivo: Determinar el fenotipo de resistencia y genes expresados en la producción de ß-lactamasas AmpC en bacilos gramnegativos de aislados clínicos en un centro hospitalario. Métodos: Estudio observacional, descriptivo y de corte transversal. Se seleccionaron 78 cepas bacterianas como portadoras de ß- lactamasas AmpC. Se les realizó prueba de aproximación de disco; a las cepas con resultado positivo se seleccionaron para extracción de ADN y PCR multiplex para detección de 6 familias genes AmpC. Se determinó la frecuencia por tipo de muestra, servicio y comparación con el perfil de susceptibilidad. Resultados: De las cepas seleccionadas con fenotipo AmpC, el 57,6 por ciento (45/78) se consideró caso confirmado ß-lactamasas AmpC por su positividad para la prueba confirmatoria. La técnica molecular utilizada confirmó en el 40 por ciento (18/45) la presencia de genes AmpC. Se obtuvo con mayor frecuencia el gen MIR n= 9 (20 por ciento), seguido de DHA n= 7 (15 por ciento). Conclusiones: La detección oportuna de genes que codifican para ß-lactamasas AmpC permite establecer estrategias para evitar la circulación mediada por plásmidos en hospitales, así como utilizar mejores opciones terapéuticas que no induzcan a otros mecanismos de resistencia(AU)

Introduction: AmpC ß--lactamases are enzymes with hydrolytic activity. They may be either constitutive or inducible. No standardized method is available for their phenotypical determination by international standards. Their detection by molecular biology could be a useful alternative for the surveillance and control of the spread of clones circulating in hospital environments. Objective: Determine the resistance phenotype and genes expressed in the production of AmpC ß-lactamases in Gram-negative bacilli from clinical isolates in a hospital. Methods: An observational descriptive cross-sectional study was conducted. A total 78 bacterial strains were selected as carriers of AmpC ß-lactamases. Disc approximation tests were performed. The strains testing positive were selected for DNA extraction and multiplex PCR for detection of six AmpC gene families. Determination was made of the frequency per sample type, service and comparison with the susceptibility profile. Results: Of the strains selected with AmpC phenotype, 57.6 percent (45/78) were considered to be AmpC β-lactamase confirmed cases, due to their positive confirmatory test. The molecular technique used confirmed the presence of AmpC genes in 40 percent (18/45) of the cases. The gene most commonly obtained was MIR n= 9 (20 percent), followed by DHA n= 7 (15 percent). Conclusions: Timely detection of genes encoding for AmpC ß-lactamases makes it possible to set up strategies to prevent plasmid-mediated circulation in hospitals, as well as apply better therapeutic options that do not induce other resistance mechanisms(AU)

Identificación molecular de Entamoeba histolytica, Entamoeba dispar y Entamoeba moshkovskii en niños con diarrea en Maracaibo, Venezuela / Molecular identification of Entamoeba histolytica, E. dispar, and E. moshkovskii in children with diarrhea from Maracaibo, Venezuela

Resumen | Introducción. Las amebas no patógenas Entamoeba dispar, Entamoeba moshkovskii y Entamoeba bangladeshi son morfológicamente idénticas a Entamoeba histolytica, parásito responsable de la amebiasis, por lo cual se necesitan técnicas moleculares para diferenciarlas. Objetivo. Determinar la frecuencia de las diferentes especies de Entamoeba mediante reacción en cadena de la polimerasa (Polymerase Chain Reaction, PCR) en muestras fecales de niños menores de cinco años con diarrea, provenientes de Maracaibo (Venezuela). Materiales y métodos. Se recolectó una muestra fecal por individuo en 75 niños con diarrea (grupo de casos) y en 25 niños sin diarrea (grupo control). Las heces se evaluaron mediante examen microscópico, método de concentración de formól-éter y PCR múltiple anidada en una sola ronda para identificar E. histolytica, E. dispar y E. moshkovskii. Además, se hizo una encuesta en la que se recopilaron los datos demográficos, signos, manifestaciones clínicas y estrato socioeconómico de los niños. Resultados. El 48 % de los participantes (38 del grupo de casos y 10 del grupo de control) tenían enteroparásitos. Solo en las muestras de cuatro de los niños, se encontraron quistes del complejo Entamoeba (tres en el grupo de casos y uno en el de control). Mediante PCR se amplificaron nueve muestras (9 %) para la detección de las amebas estudiadas. En el grupo de casos se registraron tres (28,13 %) de E. histolytica, cuatro (30,50 %) de E. dispar y una (9,37 %) de E. moshkovskii, en tanto que solo una (25 %) muestra amplificó para E. dispar en el grupo de control. Conclusión. En general, predominó E. dispar; sin embargo, todos los infectados con E. histolytica se detectaron en el grupo de niños con diarrea y se detectó el primer caso de E. moshkovskii en la región.

Abstract | Introduction: Entamoeba histolytica is an amebiasis-producing parasite. However, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba bangladeshi are non-pathogenic amoebae morphologically identical to it and, therefore, molecular techniques are required for their differentiation. Objective: To determine the frequency of Entamoeba species by polymerase chain reaction (PCR) in fecal samples from children under five years with diarrhea from Maracaibo (Venezuela). Materials and methods: A fecal sample per individual was collected from 75 children with diarrhea (case group) and 25 children without diarrhea (control group). Stools were evaluated by microscopic examination, formol-ether concentration method, and nested-multiplex PCR in a single round for the identification of E. histolytica, E. dispar, and E. moshkovskii. In addition, a survey was conducted in which demographic data, signs, clinical manifestations, and socioeconomic status were registered. Results: In total, 48% of the children (38 from the case group and 10 from the control group) had intestinal parasites. Only four children presented cysts of the Entamoeba complex in their samples (three from the case group and one from the control group). By means of PCR, nine samples (9%) amplified for the studied amoebae. In the case group, three (28.13%) amplified for E. histolytica, four (30.50%) for E. dispar, and one (9.37%) for E. moshkovskii while only one (25%) sample amplified for E. dispar in the control group. Conclusion: In general, E. dispar predominated. Nevertheless, all those infected with E. histolytica were detected within the group of children with diarrhea and we reported the first case of E. moshkovskii in the region.

Impacto del uso de PCR múltiple en diagnóstico etiológico y tratamiento de infecciones respiratorias agudas en un hospital privado del norte del país / Impact of the use of multiplex PCR on etiological diagnosis and treatment of acute respiratory infections in a private hospital of the north of the country

Resumen Antecedentes y objetivo: Las enfermedades del sistema respiratorio son causa frecuente de prescripción de antibióticos. Actualmente se emplean nuevas tecnologías para su diagnóstico como el FilmArray Respiratory Panel. El objetivo de este estudio es identificar la correlación entre el diagnóstico y tratamiento de infecciones de vías respiratorias con el resultado de PCR para virus respiratorios. Material y métodos: Estudio descriptivo, transversal, retrospectivo, se incluyeron 134 pacientes atendidos en el Hospital Christus Muguerza en Saltillo, Coahuila. Para todos los casos se analizaron los resultados del panel y el tratamiento que recibieron los pacientes. Resultados: El 58 % recibió tratamiento antibiótico a su ingreso, el 13 % tratamiento combinado (antibiótico + antiviral), 27 % recibió tratamiento sintomático y el 2 % fue tratado con antiviral de primera instancia. Posterior al resultado el 38 % continuó con antibiótico, el 30 % con antibiótico y antiviral, 13.8 % se manejó con antiviral y el 18.2 % con tratamiento sintomático. Conclusión: A pesar de la alerta mundial por la resistencia a los antimicrobianos se sigue tratando a los pacientes con antibióticos, por una situación que se cree está influenciada por varios factores.

Abstract Background and objective: Respiratory system diseases represent one of the leading cause of prescription of antibiotics. At present, new technologies for the diagnosis are being used, including the FilmArray Respiratory Panel. The objective was to identify the correlation between the diagnosis and treatment of respiratory tract infections with the result of PCR for respiratory viruses. Material and methods: Descriptive, cross-sectional, restrospective study. 134 patients were included treated at the Christus Muguerza Hospital in Saltillo, Coahuila. For all cases, the positive results of this test and the treatment patients received were analyzed. Results: 58 % received antibiotic treatment at admission, 13 % received combined treatment (antibiotic + antiviral), 27 % received symptomatic treatment since their admission and 2 % whit antiviral. After receiving a positive result for respiratory viruses, 38 % continued with antibiotics, 30 % with antibiotics and antivirals, 13.8 % only managed with antivirals and 18.2% with symptomatic treatment. Conclusion: Although we are currently on global alert for resistance to antibiotics, there is a lack of awareness about the prescription of antibiotics, due to a situation which is believed to be influenced by several factors.

Development of tetra-primer ARMS-PCR protocol to genotype the prolactin receptor SNP 39136666 and assessment of this SNP in Brazilian locally adapted cattle breeds / [Desenvolvimento de protocolo de tetra-primer ARMS- PCR para genotipagem do SNP 39136666 do receptor da prolactina e avaliação desse SNP em raças bovinas brasileiras localmente adaptadas]

As raças taurinas de origem ibérica Limonero e Carora (Bos primigenius taurus) possuem o fenótipo de pelo curto, liso e com baixa densidade folicular, o que confere a esses animais maior tolerância térmica e melhor produtividade em regiões quentes. Diferentes mutações associadas a esse fenótipo foram descritas no gene do receptor de prolactina PRLR, localizado no cromossomo bovino BTA20. Uma mutação recentemente encontrada é a substituição do nucleotídeo C por T, SNP 39136666 (p. R497*), no exon 11, que gera um códon de parada e, consequentemente, uma menor isoforma desse receptor. Neste trabalho, desenvolveu-se um protocolo rápido e de baixo custo para detecção desse SNP, utilizando-se a técnica de tetra-primer ARMS-PCR. Assim, foi possível detectar essa mutação nas raças brasileiras de origem ibérica localmente adaptadas: Caracu, Crioulo Lageano, Mocho Nacional e Pantaneiro. O alelo T foi mais frequente na raça Caracu (80%), enquanto o alelo C foi mais frequente na raça Crioulo Lageano (84%). Essa simples metodologia pode ser usada para genotipar esse SNP e ajudar na aplicação dessas informações moleculares em programas de melhoramento focados na tolerância térmica em bovinos taurinos e seus mestiços.(AU)

Detection of the main multiresistant microorganisms in the environment of a teaching veterinary hospital in Brazil / Detecção dos principais microrganismos multirresistentes no ambiente de um hospital veterinário de ensino no Brasil

Contamination of the veterinary hospital environment with multiresistant pathogens endangers not only hospitalized animals, but also the workplace safety of veterinarians and nurses, animal guardians and, when in case of a teaching hospital, veterinary students. The objective of this study was to map the main points of bacterial contamination of a veterinary teaching hospital in Brazil to identify multiresistant microorganisms and their antimicrobial resistance genes. Samples were collected from 39 different locations of a veterinary school hospital which comprised a pool according to each hospital environment. In certain environments, more than one pool has been collected. All samples were collected in quadruplicates for the selective isolation of the main multiresistant microorganisms: methicillin-resistant Staphylococcus (MRS), vancomycin resistant Enterococcus (VRE), cephalosporinases and/or extended-spectrum beta-lactamase-producing Gram-negative bacteria (ESBL) and Carbapenemase-producing (CP). After isolation and identification of isolates, multiplex-PCR reactions were performed to detect the main genes for each microorganism and antimicrobial susceptibility tests with the main antibiotics used for each bacterial group according to CLSI. Of the 39 veterinary teaching hospital sites collected, all (100%) had at least one of the microorganisms surveyed, and 17.95% (n=7) of the sites were able to isolate the four pathogens. From the 94 pools collected, it was possible to isolate MRS in 81.91% (n=77), VRE in 12.77% (n=12), cephalosporinases and/or ESBL in 62.77% (n=59) and CP in 24.47%. (n=23). Regarding MRS, the mecA gene was detected in all isolates. All isolated VREs were identified as Enterococcus faecalis and presented the vanA gene. Regarding cephalosporinases and/or ESBL, 89.83% (n=53) of the isolates presented the blaTEM gene, 57.63% (n=34) the blaOXA-1 gene, 37.29% (n=22) blaCTX-M gene from some group (1, 2, 9 ou 8/25) and 20.34% (n=12) the blaSHV gene. It was possible to identify the main microorganisms responsible for causing nosocomial infections in humans (VRE, MRS, ESBL and CP) in the veterinary hospital environment, suggesting a source of infection for professionals and students of veterinary medicine, placing a high risk for public health.(AU)

A contaminação do ambiente hospitalar veterinário com patógenos multirresistentes coloca em perigo não apenas os animais hospitalizados, mas também a segurança no local de trabalho de veterinários e enfermeiros, responsáveis por animais e, quando se tratar de um hospital de ensino, estudantes de veterinária. O objetivo deste estudo foi mapear os principais pontos de contaminação bacteriana de um hospital veterinário de ensino no Brasil, identificando microorganismos multirresistentes e seus genes de resistência antimicrobiana. As amostras foram coletadas em 39 locais diferentes de um hospital de escola veterinária, que compreendia um pool de acordo com o ambiente de cada hospital. Em certos ambientes, mais de um pool foi coletado. Todas as amostras foram coletadas em quadruplicados para o isolamento seletivo dos principais microorganismos multirresistentes: Staphylococcus resistente à meticilina (MRS), Enterococcus resistente à vancomicina (VRE), bactérias Gram-negativas produtoras de cefalosporinases e/ou beta-lactamase de espectro estendido (ESBL) e produtoras de carbapenemase (PC). Após o isolamento e identificação dos isolados, foram realizadas reações de PCR multiplex para detectar os principais genes de cada microorganismo e testes de susceptibilidade a antimicrobianos com os principais antibióticos utilizados para cada grupo bacteriano de acordo com o CLSI. Dos 39 locais do VCH coletados, todos (100%) possuíam pelo menos um dos microrganismos pesquisados e 17,95% (n=7) dos locais foram capazes de isolar os quatro patógenos. Dos 94 pools coletados, foi possível isolar MRS em 81,91% (n=77), VRE em 12,77% (n=12), ESBL em 62,77% (n=59) e carbapenemases em 24,47% (n=23). Em relação ao MRS, o gene mecA foi detectado em todos os isolados. Todos os VREs isolados foram identificados como Enterococcus faecalis e apresentaram o gene vanA. Em relação às cefalosporinases e/ou ESBL, 89,83% (n=53) dos isolados apresentaram o gene blaTEM, 57,63% (n=34) o gene blaOXA-1, 37,29% (n=22) o gene blaCTX-M de algum grupo e 20,34% (n=12) o gene blaSHV. Foi possível identificar os principais microrganismos responsáveis por causar infecções nosocomiais em humanos (VRE, MRS, ESBL e CP) no ambiente hospitalar veterinário, sugerindo uma fonte de infecção para profissionais e estudantes de medicina veterinária, colocando alto risco para a saúde pública.(AU)

Detection of Mycobacterium sp. by multiplex PCR directly from suspicious granulomas from cold chambers in the state of Bahia, Brazil

The diagnosis of bovine tuberculosis (TB) by molecular techniques has been broadly studied. These methods allow accelerating the diagnosis, in addition to presenting high specificity and sensitivity in the identification of the pathogen, critical characteristic for public health, especially when it comes to the direct diagnosis of the biologic samples, which has been little explored. This paper has evaluated a multiplex polymerase chain reaction (mPCR) as a tool to diagnose TB, which was performed directly on the granulomatous material of suspicious lesions collected in a cold chamber under state inspection in the state of Bahia, Brazil. Of the 74 samples evaluated, 14.86% were positive, with 10.81% positive for mPCR and culture, 4.05% negative for cultivation and positive for mPCR. The correlation between the cultivation and the mPCR presented agreeance higher than 61.54% of the cases. The results have indicated that the protocol proved itself effective, fast and very promising in the surveillance in slaughterhouses for the diagnosis of tuberculosis directly from the granuloma.(AU)

Rapid detection of Respiratory Pathogens using a Multiplex PCR assay among hospitalized children with Acute Respiratory Infection

Background@#Acute respiratory infection (ARI) is a major cause of morbidity and mortality among children worldwide however, local data on the etiologic diagnosis of ARI are limited. @*Objectives@#To determine the prevalence and the most commonly detected respiratory pathogens using a multiplex PCR assay, known as the Respiratory Panel, among hospitalized children with ARI and compare their clinical and laboratory differences. @*Methods@#This is a cross sectional study of children with ARI who were tested with a multiplex PCR assay. Retrospective chart review was done on these patients admitted from January 2018 to February 2020. @*Results@#There were 47 charts reviewed, mean age was 4.2 years old. Out of 47 patients, 36 (76.6%) tested positive for a pathogen. Respiratory syncytial virus (RSV) being the most common followed by Influenza A/H1-2009 and Human metapneumovirus (hMPV). Two patients had viral co-infections and no bacteria were detected on all subjects. 61.7% patients were started on antibiotics on admission. Fever and cough were the most common sign and symptom, respectively. Normal WBC (68% with neutrophilic predominance) and platelet were detected in 72.3% and 70.2% of patients, respectively; 50% of patients had normal CRP and 60.5% had abnormal findings on chest x-ray. Only the presence of chest x-ray findings was found to have a higher probability of yielding a positive Respiratory Panel p=0.27. @*Conclusion@#Among admitted patients with ARI, 76.6% tested positive for a respiratory pathogen. All were caused by viruses presenting as nonspecific manifestations – fever and cough. Clinical manifestations, CBC and CRP showed no association with the Respiratory Panel result while abnormal chest x-ray had a higher probability of yielding a positive Respiratory Panel result.