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.

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.

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.

FilmArray Meningitis/Encephalitis (ME) panel in the diagnosis of bacterial meningitis

ABSTRACT The precise diagnosis of bacterial meningitis is essential. Cytological and biochemical examination of cerebrospinal fluid (CSF) are not specific. Conventional methods for bacterial meningitis lack sensitivity or take too long for a final result. Therefore, other methods for rapid and accurate diagnosis of central nervous system infections are required. FilmArray meningitis/encephalitis (ME) panel is a PCR multiplex for simultaneous and rapid identification of 14 pathogens, including 6 bacteria, 7 viruses, and Cryptococcus. We evaluated 436 CSF samples submitted to FilmArray ME Panel. Among them, 25 cases were positive for bacteria, being Streptococcus pneumonia the most frequent (48 %). Among positive cases for bacteria, 60 % were positive only with FilmArray. All the bacterial meningitis cases in which the only positive test was FilmArray had CSF findings suggestive of bacterial meningitis, including neutrophilic pleocytosis, increased CSF protein and lactate, and decreased CSF glucose. These findings suggest that FilmArray may increase the diagnostic sensitivity for bacterial meningitis.

Detección de mutaciones causantes de distrofia muscular de Duchenne/Becker: reacción en cadena de la polimerasa Multiplex vs. Amplificación múltiple dependiente de ligación por sondas / Detection of mutations causing Duchenne and Becker muscular dystrophies: multiplex polymerase chain reaction vs. Multiplex ligation dependent probe amplification

RESUMEN Las distrofias musculares de Duchenne/Becker son enfermedades raras que reciben poca atención en nuestro medio. El objetivo del presente estudio fue implementar la técnica de amplificación múltiple dependiente de ligación por sondas (MLPA) y demostrar que tiene ventajas sobre la técnica de reacción en cadena de la polimerasa multiplex (PCR-multiplex). Se analizaron muestras de 40 individuos con diagnóstico presuntivo de distrofia muscular de Duchenne/Becker, primero por PCR-multiplex y luego por MLPA. Con la PCR-multiplex se detectaron 15 individuos con deleciones causales y con la técnica MLPA se logró diagnosticar a 21 individuos, cuatro duplicaciones y 17 deleciones. En conclusión, la técnica MLPA logra detectar mutaciones de tipo deleción y duplicación de exones, consiguiendo un mayor número de diagnósticos moleculares por alteraciones en el gen DMD.

ABSTRACT Duchenne and Becker muscular dystrophies are rare diseases that receive limited attention in our field. The objective of this study was to implement the Multiplex Ligation-dependent Probe Amplification technique (MLPA) and to demonstrate that it has advantages over the Multiplex Polymerase Chain Reaction (Multiplex PCR) technique. Samples from 40 individuals with a presumptive diagnosis of Duchenne and Becker muscular dystrophies were analyzed: first by Multiplex PCR and then by MLPA. Fifteen individuals with causal deletions were detected with Multiplex PCR, while the MLPA technique was able to diagnose 21 individuals, four duplications, and 17 deletions. In conclusion, the MLPA technique can detect mutations of the exon deletion and duplication type, yielding a larger number of molecular diagnoses due to alterations in the DMD gene.

Validation of multiplex PCR for the diagnosis of acute bacterial meningitis in culture negative cerebrospinal fluid / Validação do PCR multiplex para o diagnóstico de meningite bacteriana aguda com cultura de liquido cefalorraquidiano negativa

ABSTRACT This study evaluated the operational characteristics of the multiplex polymerase chain reaction (PCR) for cerebrospinal fluid (CSF) from patients with cellular and biochemical characteristics of acute bacterial meningitis and positive or negative CSF cultures. Methods: Multiplex PCR was performed for 36 CSF samples: culture-proven acute bacterial meningitis (n = 7), culture-negative acute bacterial meningitis (n = 17), lymphocytic meningitis (n = 8), and normal CSF (n = 4). The operational characteristics of multiplex PCR were evaluated with definite and probable bacterial meningitis, using culture positive, cytological and biochemical CSF characteristics as the gold standard. Results: Multiplex PCR for CSF was efficient in the group with CSF cellular and biochemical characteristics of acute bacterial meningitis but with a negative CSF culture. This group demonstrated high specificity, positive predictive value, and efficiency. Conclusions: Multiplex PCR for CSF can improve the speed and accuracy of acute bacterial meningitis diagnosis in a clinical setting as a complement to classical immunological and bacteriological assays in CSF. It is also useful for CSF culture-negative acute bacterial meningitis.

RESUMO Este estudo avaliou as características funcionais da reação em cadeia da polimerase (PCR) multiplex para amostras de líquido cefalorraquidiano (LCR) de pacientes com características celulares e bioquímicas de meningite bacteriana aguda e culturas de LCR positivas ou negativas. Métodos: O PCR multiplex foi realizado em 36 amostras de LCR: meningite bacteriana aguda comprovada por cultura (n = 7), meningite bacteriana aguda com cultura negativa (n = 17), meningite linfocítica (n = 8) e LCR normal (n = 4). As características funcionais do PCR multiplex foram avaliadas para meningite bacteriana definitiva e provável, utilizando cultura positiva, características citológicas e bioquímicas do LCR como padrão-ouro. Resultados: O PCR multiplex do LCR foi eficiente no grupo com características celulares e bioquímicas do LCR de meningite bacteriana, mas com cultura do LCR negativa. Este grupo demonstrou especificidade, valor preditivo positivo e eficiência altos. Conclusões: Os autores concluíram que o PCR multiplex do LCR pode melhorar a velocidade e a precisão do diagnóstico de meningite bacteriana em um ambiente clínico como complemento aos ensaios imunológicos e bacteriológicos clássicos no LCR. Também é útil para meningite bacteriana aguda com cultura de LCR negativa.

Streptococcus agalactiae - Distribuição sorotípica e relação com fatores de virulência e resistência antimicrobiana / Streptococcus agalactiae - Serotype distriburion and correlation with virulence factors and antibiotic resistance

Streptococcus agalactiae, ou Estreptococo do Grupo B, é um microrganismo que encontrado na microbiota intestinal, vaginal e/ou geniturinária de 10-30% de mulheres saudáveis. A principal infecção causada por S. agalactiae é a sepse neonatal. O bebê pode adquirir o microrganismo durante o parto ao passar pelo canal vaginal, ou até mesmo durante a gestação, caso haja ascensão de S. agalactiae para o útero. Existem diversos fatores associados à infecção do feto por S. agalactiae quando a mãe é colonizada, tais como fator CAMP, cápsula de polissacarídeos, hialuronidase, ß-citolisina/hemolisina e pili. Não existe consenso ou recomendação técnica sobre o tema no Brasil. Segundo o Caderno de Atenção Básica ao Pré-Natal, não existem estudos que levem à recomendação da antibioticoterapia intraparto. É necessário elucidar as características genotípicas de cepas de S. agalactiae isoladas no Brasil para alinhar as práticas clínicas às características fenotípicas do microrganismo. Desta forma, os objetivos deste projeto são: i) classificar cepas de S. agalactiae isoladas de gestantes e não gestantes quanto ao sorotipo capsular, por PCR Multiplex, ii) avaliar a presença e distribuição de fatores de virulência, por PCR e iii) avaliar o perfil de resistência antimicrobiana, pelo método de disco difusão e teste D. Os achados de virulência e resistência a antimicrobianos foram comparados com os sorotipos, gestação, localização geográfica e sítio de isolamento. Foram analisadas 292 cepas isoladas de gestantes e não gestantes em São Paulo, São José dos Campos e Rio de Janeiro. O sorotipo Ia foi o mais prevalente entre as cepas. Na cidade de São José dos Campos não houve diferença significativa entre a prevalência dos sorotipos Ia e V, sendo que o sorotipo V foi mais abundante do que nas cidades de São Paulo e Rio de Janeiro. O sorotipo II foi mais abundante em mulheres não gestantes do que gestantes. Não foram encontradas cepas resistentes à Penicilina e vancomicina; contudo a resistência a Cefepima, Eritromicina e Clindamicina ficou em torno de 22%. Foram encontradas diferenças entre os sorotipos quanto à resistência, genes de virulência e sítio de isolamento das cepas. Portanto essas diferenças podem se refletir no perfil epidemiológico da infecção por S. agalactiae quanto à localização geográfica também quanto à gestação. A incidência de sepse causada por S. agalactiae diminuiu muito nas últimas décadas, contudo o monitoramento constante é necessário para alinhar as práticas clínicas às características fenotípicas do microrganismo

Streptococcus agalactiae, or Group B Streptococcus, is a microorganism found in intestinal, vaginal and/or genitourinary microbiota from about 10-30% of all healthy women. The main infection caused by S. agalactiae is neonatal sepsis. The baby can contract the infection during labor when passing through the vaginal canal, or even during pregnancy, if S. agalactiae ascends from the vaginal canal to the uterus. There are several factors associated to the infection of the fetus by S. agalactiae when the mother is colonized, such as the CAMP factor, polysaccharide capsule, hyaluronidase, ß-cytolysin/hemolysin and pili. There is no consensus or technical recommendation regarding this theme in Brazil. According to the Brazilian guidelines to prenatal care, there is no research that justifies the implementation of intrapartum antibiotic therapy. There is a need to clarify genotype characteristics of S. agalactiae strains isolated in Brazil in order to align clinical practices to phenotypical characteristics of this microorganism. This way, the goals of this project are: i) to classify S. agalactiae strains isolated from pregnant and nonpregnant women according to their capsular serotype, using PCR Multiplex, ii) to evaluate the presence and distribution of virulence factors, using PCR and iii) to evaluate their antibiotic resistance profile, using disk-diffusion and D-zone tests. The findings regarding virulence and resistance were compared to serotypes, pregnancy, geographic localization and the site where the sample was isolated. A total of 292 strains from pregnant and nonpregnant women from the cities of São Paulo, São José dos Campos and Rio de Janeiro were analyzed. Serotype Ia was the most prevalent among the strains. In São José dos Campos there was no significate difference in the prevalence of serotypes Ia and V. Serotype V was the most abundant in São Paulo and Rio de Janeiro. Serotype II was most prevalent in nonpregnant women when compared to pregnant women. No resistance to Penicillin nor Vancomycin was found. However, resistance to Cefepime, Erythromycin or Clindamycin was found in around 22% of strains. There were differences among serotypes regarding resistance, virulence genes and site where the strain was isolated. Therefore these differences can reflect into the epidemiologic profile of S. agalactiae infection in regards to geographic localization and pregnancy. The incidence of sepsis caused by S. agalactiae has decrease in the last few decades, however constant monitoring is necessary in order to align clinical practice to the microorganisms phenotypical characteristics

Presencia de Streptococcus Mutans Genotipo C en niños y adolescentes peruanos con caries / Presence of Streptococcus Mutans Genotype C in Peruvian Children and Adolescents with Caries

RESUMEN La caries es una de las enfermedades de naturaleza infecciosa, crónica transmisible muy prevalente en el Perú, relacionada a la presencia del Streptococcus mutans, los hábitos de higiene y nutricionales. Objetivo: El propósito de este estudio fue determinar la presencia del genotipo C en el Streptococcus mutans en niños y adolescentes peruanos, utilizando la técnica PCR- Multiplex; y su asociación con la prevalencia de caries dental. Material y método: Se trabajó con una muestra de 78 niños y adolescentes de ambos sexos de Lima. El estudio consistió en dos fases, en la primera se obtuvo la saliva estimulada, para el cultivo bacteriano, las mismas que fueron sembradas en agar Mitis Salivarius con bacitracina y sulfisoxasol. En la segunda fase se realizó la genotipificación de acuerdo con su perfil enzimático. Para la extracción de ADN se utilizó el GF-1 Bacterial DNA Extraction Kit de GeneONGmbH para lo cual se realizó cultivos de las cepas de Streptococcus sp en el caldo BHI con sacarosa a 37ºC por 24 horas. Resultados: Se evidencia la presencia de Streptococcus mutans en 75.6%: 59 de 78 muestras de saliva. Los resultados de la genotipificación por PCR Multiplex demuestran la presencia de 22 muestras de saliva de Streptococcus mutans con genotipos C (37,29%) y 37 muestras (62,71%) que no pertenecen a dicho Genotipo. Conclusiones: Los resultados evidenciaron que el Streptococcus mutans genotipo C no está relacionado al sexo, grupo etario ni a la presencia de caries dental.

ABSTRACT Caries is one of the diseases of infectious nature, chronic transmissible very prevalent in Peru, related to the presence of Streptococcus mutans, hygienic and nutritional habits. Objective: The purpose of this study was to determine the presence of genotype C in Streptococcus mutans in Peruvian children and adolescents, using the PCR-Multiplex technique; and its association with the prevalence of dental caries. Materials and methods: The study was done with a sample of 78 children and adolescents of both sexes from Lima. The study consisted of two phases, on the first one the stimulated saliva was obtained, for the bacterial culture, the same ones that were grown on Mitis Salivarius agar with bacitracin and sulfisoxasol. On the second phase, genotyping was carried out according to its enzymatic profile. For the extraction of DNA, the Gene Extraction Kit GG-1 Bacterial DNA was used, for which cultures of Streptococcus sp strains were performed in the BHI broth with sucrose at 37ºC for 24 hours. Results: The presence of Streptococcus mutans was evidenced in 59 (75.6%) of 78 saliva samples. The results of the genotyping by PCR Multiplex demonstrate the presence of 22 saliva samples of Streptococcus mutans with genotypes C (37,297%) and 37 samples 62, 71 % without this Genotype. Conclusions: The results showed that the presence of genotype C is not related to sex, age group or the presence of dental caries.

Cytogenomic assessment of the diagnosis of 93 patients with developmental delay and multiple congenital abnormalities: The Brazilian experience

OBJECTIVE: The human genome contains several types of variations, such as copy number variations, that can generate specific clinical abnormalities. Different techniques are used to detect these changes, and obtaining an unequivocal diagnosis is important to understand the physiopathology of the diseases. The objective of this study was to assess the diagnostic capacity of multiplex ligation-dependent probe amplification and array techniques for etiologic diagnosis of syndromic patients. METHODS: We analyzed 93 patients with developmental delay and multiple congenital abnormalities using multiplex ligation-dependent probe amplifications and arrays. RESULTS: Multiplex ligation-dependent probe amplification using different kits revealed several changes in approximately 33.3% of patients. The use of arrays with different platforms showed an approximately 53.75% detection rate for at least one pathogenic change and a 46.25% detection rate for patients with benign changes. A concomitant assessment of the two techniques showed an approximately 97.8% rate of concordance, although the results were not the same in all cases. In contrast with the array results, the MLPA technique detected ∼70.6% of pathogenic changes. CONCLUSION: The obtained results corroborated data reported in the literature, but the overall detection rate was higher than the rates previously reported, due in part to the criteria used to select patients. Although arrays are the most efficient tool for diagnosis, they are not always suitable as a first-line diagnostic approach because of their high cost for large-scale use in developing countries. Thus, clinical and laboratory interactions with skilled technicians are required to target patients for the most effective and beneficial molecular diagnosis.

Precisión diagnóstica de tres pruebas moleculares para detectar la tuberculosis multirresistente / Diagnostic accuracy of three technologies for the diagnosis of multi-drug resistant tuberculosis

Resumen Introducción. La tuberculosis multirresistente (TB-MDR) y la extremadamente resistente (TB-XDR) constituyen un problema de salud pública a nivel mundial. Su detección oportuna permitiría reducir la carga de la enfermedad y su impacto económico en los sistemas de salud. Objetivo. Revisar sistemáticamente la información relacionada con la precisión diagnóstica de tres pruebas moleculares para detectar la tuberculosis multirresistente y la extremadamente resistente. Materiales y métodos. Se hizo una revisión sistemática de la literatura, según los lineamientos de Cochrane, de los estudios en población inmunocompetente relacionados con la precisión diagnóstica de tres pruebas moleculares para detectar la tuberculosis multirresistente y la extremadamente resistente. La búsqueda de los estudios publicados a partir del 2007 se hizo en Medline y Embase. La precisión diagnóstica de las pruebas se estableció con base en los valores máximos y mínimos de sensibilidad y especificidad, y en los valores predictivos positivos y negativos. Resultados. Se detectaron ocho estudios sobre la precisión diagnóstica de la prueba GeneXpert MTB/RIF(r), 12 sobre la de GenoType MTBDRplus(r) y 13 sobre la de GenoType MTBDRsl(r). La especificidad de GeneXpert MTB/RIF(r) osciló entre 91 y 100 % y su sensibilidad, entre 33,3 y 100 %. La sensibilidad de GenoType MTBDRplus(r) varió entre 82 y 100 %, en tanto que la sensibilidad y la especificidad de GenoType(r) MTBDRsl fluctuaron entre 56 y 100 % y 21 y 100 %, respectivamente. Conclusión. Según los estudios consultados, los tres métodos de diagnóstico evaluados presentabanuna adecuada eficacia diagnóstica para detectar la tuberculosis multirresistente y la extremadamente resistente.

Abstract Introduction: Multi-drug resistant (MDR-TB) and extensively drug-resistant (XDR-TB) tuberculoses are a global public health problem. Their timely detection might reduce the burden of the disease and the economic impact on health systems worldwide. Objective: To conduct a literature review of the diagnostic accuracy of three molecular tests to detect multi-drug resistant and extensively drug-resistant tuberculoses. Materials and methods: A systematic literature review following Cochrane methodology was carried out to study the diagnostic accuracy of three molecular tests to detect MDR-TB and XDR-TB in previous studies among immunocompetent population. Articles indexed in Medline and Embase were reviewed starting in 2007. Diagnostic accuracy was reported by sensitivity, specificity, and positive and negative predictive values of each test. Results: In total, 8, 12 and 13 studies were included to assess the diagnostic accuracy of GeneXpert MTB/RIF(r), GenoType MTBDRplus (r) and GenoType MTBDRsl (r), respectively. The specificity of GeneXpert MTB/RIF(r) ranged between 91 and 100%, and its sensitivity between 33.3 and 100%. The sensitivity of GenoType(r) MTBDRplus (r) ranged between 88 and 100%. The sensitivity and specificity of GenoType MTBDRsl (r) to evaluate drug resistance ranged between 56 and 100% and 21 and 100%, respectively. Conclusion: The three diagnostic tests evaluated have shown an adequate diagnostic accuracy to detect MDR and XDR tuberculoses.

Sixty-six Biallelic Genetic Markers on Y chromosome by MALDI-TOF-MS / 法医学杂志

OBJECTIVES@#To analyse the genetic polymorphisms of 66 biallelic genetic markers on Y chromosome in Eastern Chinese Han population, and evaluate their values in forensic application.@*METHODS@#Genotyping of 66 biallelic genetic markers on Y chromosome was studied in 205 unrelated males of Eastern Chinese Han population by multiplex PCR combined matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）. The allele frequencies on the loci to be tested were calculated by direct counting method, and the gene diversity （GD） and haplotype diversity （HD） were calculated by corresponding formulas. The haplotypes of this system were tested by software Arlequin v3.5.2.2 and the comparison of population genetics were analyzed.@*RESULTS@#A total of 60 biallelic genetic markers on Y chromosome were polymorphic in males of Eastern Chinese Han population, and the ranges of GD were from 0.038 5 to 0.501 9. Eighty-five different haplotypes were observed and the HD was 0.970 3. The differences of partial SNP loci between the Han population of Eastern China and that of Xinjiang and Guangdong were statistically significance.@*CONCLUSIONS@#Sixty biallelic genetic markers and the detection system can complementally provide genetic information in kinship testing and individual identification. The MALDI-TOF-MS technology is able to type biallelic genetic markers.

Técnica de PCR-multiplex como método diagnóstico de infecciones de transmisión sexual / Multiplex polymerase chain reaction as diagnostic method of sexually transmitted diseases

Introducción: las Infecciones de Transmisión Sexual afectan a personas de cualquier etnia, estrato social y edad. Son más comunes en quienes mantienen conductas sexuales riesgosas. El diagnóstico convencional solo permite determinar un patógeno a la vez y no se realiza una determinación completa de los microorganismos causantes de estas infecciones. El uso de nuevos y mejores métodos de diagnóstico como son los métodos moleculares, constituye una herramienta de investigación oportuna que se ajusta a la realidad actual. Objetivo: determinar los agentes patógenos más frecuentes en las infecciones de trasmisión sexual diagnosticados por reacción en cadena de la polimerasa-multiplex en todas las mujeres que acuden al centro de salud No. 1 de Azogues. Métodos: se realiza un estudio retrospectivo en mujeres que acuden al centro de salud No. 1 de Azogues, desde septiembre de 2015 hasta marzo del 2016. Resultados: de las mujeres que acudieron a consulta, 46 por ciento tenían entre 34 y 44 años de edad. Las de procedencia urbana representaban el 66 por ciento y las que tenían un nivel de escolaridad superior, representaban 38 por ciento. La técnica de PCR Multiplex permitió determinar la presencia de Mycosplasma hominis, Neisseria gonorrhoeae, Ureoplasma urealyticun y trichomonas vaginalis aun cuando 98 por ciento de las pacientes estaba asintomática. Entre los factores de riego para la enfermedad se encontró no utilizar preservativo y conocimiento insuficiente de las infecciones de transmisión sexual y sus síntomas. Conclusiones: la técnica de PCR-Multiplex constituye una herramienta eficaz para la detección precoz de varios agentes patógenos(AU)

Introduction: Sexually transmitted diseases affect people from any ethnics, social strata and age. They are more frequent in people with risky sexual behaviors. The conventional diagnosis only allows determining a pathogen at a time and the causative microorganisms of these infections are not fully identified. The use of new better methods of diagnosis such as the molecular methods is a timely research tool that suits to the present realities. Objective: To determine the most common pathogenic agents in sexually transmitted diseases that are diagnosed through multiplex-polymerase chain reaction in all women who go to the health center no.1 in Azogues. Methods: Retrospective study of women who go to the health center no.1 in Azogues from September 2015 to March 2016. Results: Of the group of women who went to physician´s office, 46 percent were 34 to 44 years. Women living in urban places accounted for 66 percent and those with higher education 38 percent. Multiplex polymerase chain reaction allowed determining the presence of Mycoplasma hominis, Neisseria gonorrhoeae, Ureoplasma urealyticun and trichomonas vaginalis even when 98 percent of patients were asymptomatic. Among the risk factors of the disease were non use of condom and lack of knowledge of sexually transmitted infections and their symptoms. Conclusions: Multiplex polymerase chain reaction technique is an effective tool for early detection of several pathogenic agents(AU)

Isolamento de Vibrio spp de camarões comercializados in natura na cidade de São Gonçalo - RJ / Isolation of Vibrio spp from shrimp commercialized in natura in the city of Sao Gonçalo - RJ

Bactérias do gênero Vibrio fazem parte da microbiota de camarões, pois têm capacidade de associar-se à quitina presente no exoesqueleto destes animais e ao zooplancton, que por sua vez são consumidos por estes animais. O gênero contém pelo menos 12 espécies patogênicas, incluindo V. cholerae, responsável por várias pandemias de cólera. A contaminação humana acontece através do consumo de alimentos, principalmente de origem marinha, crus ou mal cozidos. Por se tratar de um tipo de pescado amplamente consumido pela população, este trabalho teve como objetivo investigar a presença de espécies de Vibrio em camarões comercializados in natura na cidade de São Gonçalo-RJ. Os camarões foram adquiridos em duas peixarias da cidade e caracterizados por metodologia convencional e molecular; cento e vinte e nove amostras testaram positivamente para as provas bioquímicas realizadas e, destas, cinquenta e duas testaram positivamente para os testes moleculares. Visando investigar a identidade das espécies de Vibrio, as amostras foram submetidas ao PCR multiplex para 4 espécies (V. cholerae, V. mimicus, V. parahaemolyticus, V. vulnificus). Doze isolados foram identificados como V. parahaemolyticus e 9 como V.cholerae não O1. Dentre os demais isolados, 31 demonstram se tratar de outras espécies de Vibrio spp. O sítio com o maior número de isolados foi a casca, seguida pelo hepatopâncreas/ hemolinfa. A ribotipagem por PCR das 21 cepas demonstrou claramente a separação das cepas de V.parahaemolyticus e V.cholerae. As cepas de V. cholerae e V. parahaemolyticus demonstraram alto índice de resistência a ampicilina (83,33%) e 100% de sensibilidade à nitroflurantoína e tetraciclina. Sete cepas (38,8%) apresentaram perfil de multirresistência a dois antimicrobianos. Nossos resultados demonstram a presença de espécies patogênicas de Vibrio em amostras de pescados amplamente consumidos pela população.

Establishment and Verification of 6-color Fluorescent-labeled Rapid PCR Amplification System / 法医学杂志

OBJECTIVE@#To establish the rapid PCR amplification program and system and to verify the technical indexes.@*METHODS@#PCR multiplex and capillary electrophoresis detection of 24 autosomal STR loci and one Y-STR loci using the 6-color fluorescence marking technology, as well as A melogenin and Y-InDel. Meanwhile, sensitivity, specificity, identity, stability, mixing and a batch of sample tests were investigated, and the genotype of various routine samples and degraded, exfoliated cell samples were observed.@*RESULTS@#The sensitivity of the system was 0.062 5 ng. In addition, the genotype could be detected accurately only around 65 min via rapid amplification. The species-specificity was high and the genotyping of all kinds of dry blood specimens of filter paper and mixed, degraded, exfoliated cell samples were accurate.@*CONCLUSION@#The rapid amplification system can significantly improve the detection rate, and obtain accurate and stable genotyping results, which may be important implications for the establishment of STR database and study on population genetics and forensic identification.

Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis

PURPOSE: Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay. MATERIALS AND METHODS: We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR. RESULTS: Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard. CONCLUSION: These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.

Establishing a 29 Y-STR Loci Multiplex PCR System / 法医学杂志

OBJECTIVE@#To establish a 29 Y-STR loci multiplex PCR system for investigating the genetic polymorphisms and to assess its application value in forensic science.@*METHODS@#A multiplex PCR system was established using a five color fluorescence labeling 29 Y-STR loci (DYS456, DYS389 I , DYS437, DYS447, DYS389 11, DYS438, DYS522, DYS460, DYS458, DYS622, DYS390, DYS392, DYS448, DYS449, DYS391, Y-GA TA-H4, DYS388, DYS19, DYS385a/b, DYS527a/b, DYS393, DYS459a/b, DYS635, DYS439, DYS570 and DYS627) for multiple amplification and capillary electrophoresis. And its applicability was validated with genetic polymorphism data of 29 Y-STR of unrelated 2,000 male samples in Shandong Han population.@*RESULTS@#A total of 1,981 different haplotypes of 2,000 individuals showed genotype diver- sity between 0.370 0 and 0.965 4. The system provided stable and accurate typing with high sensitivity of 0.05 ng. It satisfied the needs of variety of routine biological samples.@*CONCLUSION@#The 29 Y-STR loci multiplex PCR system could be applied for actual cases and establishment of Y-STR database. In addition, it has great significance in forensic science practices and related research.

Establishment of a 15 loci multiplex amplification system and the genetic poly- morphism in Xinjiang Uygur population / 法医学杂志

OBJECTIVE@#To develop a five fluorescence-labeled multiplex amplification system for 15 loci and study genetic polymorphism in Xinjiang Uygur population.@*METHODS@#The STR loci were screened. The alleles were named according to the number of repeats by sequencing. The sensitivity, species specificity, identity and stability of the five fluorescence-labeled multiplex amplification system for the 15 loci were all tested. Then, the genetic polymorphism was analyzed in Xinjiang Uygur population and compared with other ethnic groups including Xizang Tibetan, Xiuyan Manchu, and Guangzhou Han population.@*RESULTS@#The 15 loci multiplex amplification system was established. The sensitivity was 0.3 ng with good species specificity, identity and stability. The distributions of genotype for 13 STR loci in Uygur population were in accordance with Hardy-Weinberg equilibrium with no genetic linkage between these loci. Most loci showed statistically significant among different populations.@*CONCLUSION@#The established system has application value in forensic evidence. The 13 STR loci in Uygur population have

Xenomonitoring of Different Filarial Nematodes Using Single and Multiplex PCR in Mosquitoes from Assiut Governorate, Egypt

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.

Xenomonitoring of Different Filarial Nematodes Using Single and Multiplex PCR in Mosquitoes from Assiut Governorate, Egypt

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.