¿La detección de toxinas de Clostridioides difficile es necesaria cuando se detecta la enzima glutamato deshidrogenasa? / Is Clostridioides difficile toxins detection necessary when the glutamate dehydrogenase enzyme is detected?

Resumen Introducción: Clostridioides difficile causa diarrea y colitis pseudomembranosa. Su diagnóstico se realiza con la detección de glutamato-deshidrogenasa (GDH) o las toxinas A y B y se confirma con pruebas de amplificación de ácidos nucleicos. Objetivo: Definir si la determinación de GDH es redundante a la de las toxinas. Métodos: Estudio observacional retrospectivo de muestras fecales de pacientes con sospecha de infección por Clostridioides difficile. Las toxinas y GDH se determinaron mediante inmunocromatografía. Se realizó una simulación bayesiana con los cocientes de probabilidad; se consideró significativo un valor de p < 0.05. Resultados: Se analizaron 329 resultados de GDH y toxinas A y B. Se encontró una prevalencia de infección de Clostridioides difficile de 18.2 %. La sensibilidad y especificidad de la prueba de GDH fue de 0.90 y 0.89, respectivamente. El cociente de probabilidad positivo fue de 8.9 y el negativo, de 0.11. Conclusiones: Un resultado negativo de GDH disminuye considerablemente la probabilidad de infección, pero no la descarta. La detección de toxinas de Clostridioides difficile puede ser necesaria en instituciones donde la amplificación de ácidos nucleicos no es económica o accesible.

Abstract Introduction: Clostridioides difficile causes diarrhea and pseudomembranous colitis. Its diagnosis is made with glutamate dehydrogenase (GDH) or toxins A and B detection and is confirmed with nucleic acid amplification tests. Objective: To define if GDH determination is redundant to that of toxins. Methods: Retrospective, observational study in diarrheal stools of patients with suspected Clostridioides difficile infection. Toxins and GDH were determined by immunochromatography. Bayesian simulation was performed with likelihood ratios; a p-value < 0.05 was regarded as significant. Results: 329 GDH and toxin A and B results were analyzed. Clostridioides difficile infection prevalence was 18.2 %. Sensitivity and specificity of the GDH test were 0.90 and 0.89, respectively. Positive likelihood ratio was 8.9, and negative was 0.11. Conclusions: A negative GDH result considerably reduces the probability of infection but does not rule it out. Clostridioides difficile toxins detection may be necessary in institutions where nucleic acid amplification is not affordable or accessible.

Micetismos: Parte 3: Síndromes tempranos gastrointestinales / Mycetisms Part 3: Early Gastrointestinal Syndromes

En esta Parte 3 de la serie de cuatro artículos sobre micetismos se analizan los síndromes tempranos con síntomas gastrointestinales que se caracterizan por presentar un período de latencia muy corto, de menos de 6 horas después de la ingestión de los macromicetos. Los restantes síndromes tempranos con sintomatología compleja serán tratados en la Parte 4 de la serie. Actualmente se conocen más de 200 especies responsables de síndromes gastrointestinales, pero en este trabajo se abordarán solamente diez ejemplos que involucran los géneros Boletus [Boletus satanas (o Rubroboletus satanas) y Boletus venenatus (o Neoboletus venenata)], Hypholoma, Agaricus (Agaricus xanthodermus), Omphalotus, Lactarius, Russula, Entoloma, Chlorophyllum (Chlorophyllum molybdetes) y Leucoprinus (Leucoprinus birnbaumii). Las toxinas involucradas en estos casos presentan gran variedad estructural, desde proteínas hasta terpenoides, en particular sesquiterpenoides y triterpenoides, vinilglicina, fenol y azocompuestos, pero todas generan la misma sintomatología. Estas sustancias y otros componentes químicos de los hongos suelen ser indigestos, con una susceptibilidad variable entre los consumidores. El tratamiento es de apoyo y es estrictamente para esos casos con cuadros más graves de deshidratación. Normalmente, los casos evolucionan favorablemente después de 12 a 48 horas. Se analizan los síntomas, las toxinas involucradas, los mecanismos de acción, cuando se conocen y las especies causantes de los micetismos.

This part 3 of the series of four articles on mushroom poisoning refers to early-onset gastrointestinal syndromes, which are characterized by a very short latency period of less than 6 hours after mushroom ingestion. The remaining early-onset syndromes with complex symptoms will be treated in Part 4 of the series. Currently, more than 200 species responsible for gastrointestinal syndromes are known, but in this paper only ten examples will be addressed involving the genera Boletus [e.g., Boletus satanas (or Rubroboletus satanas), and Boletus venenatus (or Neoboletus venenata)], Hypholoma, Agaricus (e.g., Agaricus xanthodermus), Omphalotus, Lactarius, Russula, Entoloma, Chlorophyllum (e.g., Chlorophyllum molybdetes), and Leucoprinus (e.g., Leucoprinus birnbaumii). The toxins involved in these cases have a great structural variety, from proteins to terpenoids, in particular sesquiterpenoids and triterpenoids, vinylglycine, phenol, and azocompounds, but all show the same symptoms. These substances and other mushroom chemical constituents are usually indigestible, with varying consumer susceptibility. The treatment is supportive and is strictly for those cases with more severe dehydration. Usually, the cases progress favourably after 12 to 48 hours.The symptoms, toxins involved, mechanisms of action when known, and the species of mushrooms responsible for the mycetisms are analysed.

Nesta parte 3 da série de quatro artigos sobre intoxicação por cogumelos são analisadas as síndromes precoces com sintomas gastrointestinais que se caracterizam por apresentar um período de latência muito curto, de menos de 6 horas, após a ingestão de cogumelos. As síndromes precoces restantes com sintomatologia complexa serão tratadas na Parte 4 da série. Atualmente, são conhecidas mais de 200 espécies responsáveis por síndromes gastrointestinais, mas neste trabalho serão abordados apenas dez exemplos que envolvem os gêneros Boletus [Boletus satanas (ou Rubroboletus satanas) e Boletus venenatus (ou Neoboletus venenata)], Hypholoma, Agaricus (Agaricus xanthodermus), Omphalotus, Lactarius, Russula, Entoloma, Chlorophyllum (Chlorophyllum molybdetes) e Leucoprinus (Leucoprinus birnbaumii). As toxinas envolvidas nestes casos têm uma grande variedade estrutural, desde proteínas até terpenóides, em particular sesquiterpenóides e triterpenóides, vinilglicina, fenol e azo compostos, mas todas apresentam a mesma sintomatologia. Essas substâncias e outros constituintes químicos dos cogumelos costumam ser indigestos, com uma suscetibilidade variável entre aqueles que os consomem. O tratamento é de suporte e é rigorosamente para esses casos com quadros mais graves de desidratação. Normalmente, os casos evoluem favoravelmente após 12 a 48 horas. São analisados os sintomas, as toxinas envolvidas, os mecanismos de ação, quando conhecidos, e as espécies de cogumelos responsáveis pelas intoxicações.

Detection of toxigenic Clostridioides --#1;Clostridium--#3; difficile: Usefulness of two commercially available enzyme immunoassays and a PCR assay on stool samples and stool isolates / Detección de Clostridioides --#1;Clostridium--#3; difficile toxigénico: utilidad de dos métodos de enzimoinmunoensayo comerciales y una PCR en las muestras de materia fecal y en los respectivos aislamientos

The best laboratory diagnostic approach to detect Clostridioides --#1;Clostridium--#3; difficile infection (CDI) is a subject of ongoing debate. With the aim of evaluating four laboratory diagnostic methods, 250 unformed stools from patients with suspected CDI submitted to nine medical center laboratories from November 2010 to December 2011, were studied using: (1) an immunochromatographic rapid assay test that combines the qualitative determination of glutamate dehydrogenase (GDH) plus toxins A and B (QAB), the CDIFF QUIK CHEK COMPLETE assay; (2) an enzyme immunoassay for qualitative determination of toxins A and B, the RIDASCREENTC. difficile Toxin A/B assay (RAB); (3) a PCR for the toxin B gene assay (PCR); and (4) the toxigenic culture (TC).C. difficile isolates from direct toxin negative stools by QAB, RAB and PCR were evaluated for toxigenicity by the same direct tests, in order to assess the contribution of the TC (QAB-TC, RAB-TC, PCR-TC). A combination of the cell culture cytotoxicity neutralization assay (CCCNA) in stools, and the same assay on isolates from direct negative samples (CCCNA-TC) was considered the reference method (CCCNA/CCCNA-TC). Of the 250 stools tested, 107 (42.8%) were positive by CCCNA/CCCNA-TC. The GDH and PCR/PCR-TC assays were the most sensitive, 91.59% and 87.62%, respectively. The QAB, RAB, QAB/QAB-TC and RAB/RAB-TC had the highest specificities, ca. 95%. A negative GDH result would rule out CDI, however, its low positive likelihood ratio (PLR) of 3.97 indicates that a positive result should always be complemented with the detection of toxins. If the RAB, QAB, and PCR assays do not detect toxins from direct feces, the toxigenic culture should be performed. In view of our results, the most accurate and reliable methods to be applied in a clinical microbiology laboratory were the QAB/QAB-TC, and RAB/RAB-TC, with PLRs >10 and negative likelihood ratios <0.30.

El mejor procedimiento para realizar el diagnóstico de laboratorio de la infección causada por Clostridioides --#1;Clostridium--#3; difficile (ICD) es aún objeto de debate. Con el fin de evaluar cuatro métodos diagnósticos de laboratorio, se estudiaron 250 muestras de heces diarreicas provenientes de pacientes con sospecha de ICD remitidas a los laboratorios de nueve centros médicos entre noviembre de 2010 y diciembre de 2011. Dichas muestras se analizaron mediante los siguientes métodos:1) un ensayo rápido inmunocromatográfico que combina la detección cualitativa de la glutamato deshidrogenasa (GDH) y de las toxinas Ay B (QAB), CDIFF QUIK CHEK COMPLETE;2) un enzimoinmunoanálisis para la determinación cualitativa de las toxinas A/B, RIDASCREENTC. difficile Toxin A/B (RAB);3) un método molecular basado en PCR para la detección del gen que codifica la toxina B (PCR) y 4) el cultivo toxigénico (TC). Como método de referencia se utilizó la combinación del ensayo de citotoxicidad sobre cultivo de células con la neutralización de toxina mediante anticuerpo específico en los filtrados de las heces (CCCNA) y el mismo método en sobrenadantes de aislamientos de C. difficile (CCCNA-TC). La toxigenicidad de las cepas aisladas de muestras directas negativas con QAB, RAB y PCR se evaluó con los mismos métodos, con el propósito de detectar la contribución del TC (QAB-TC, RAB-TC, PCR-TC). De las 250 muestras estudiadas, 107 (42,8%) fueron positivas por CCCNA/CCCNA-TC. Los métodos GDH y PCR/PCR-TC fueron los más sensibles: 91,59 y 87,62%, respectivamente. Los métodos QAB, RAB, QAB/QAB-TC y RAB/RAB-TC mostraron las mayores especificidades, del 95%, aproximadamente. Un resultado negativo para GDH excluiría la ICD, pero su baja razón de verosimilitud positiva (PLR), que fue 3,97, indica que un resultado positivo debe complementarse con la detección de toxinas. Cuando no se detectan toxinas directas por RAB, QAB ni PCR, debería realizarse el TC. De acuerdo con nuestros resultados, los métodos más precisos y confiables para ser aplicados en un laboratorio de microbiología clínica son QAB/QAB-TC y RAB/RAB-TC, con una PLR> 10 y una razón de verosimilitud negativa < 0,30.

Características de la infección por Clostridium difficile en una institución de alta complejidad y reporte de la circulación en Colombia de la cepa hipervirulenta NAP1/027 / Characteristics of Clostridium difficile infection in a high complexity hospital and report of the circulation of the NAP1/027 hypervirulent strain in Colombia

Resumen Introducción. Clostridium difficile es el principal responsable de la diarrea asociada al uso de antibióticos. En Colombia y en Latinoamérica, el conocimiento sobre el comportamiento epidemiológico de la infección por C. difficile todavía es limitado. Objetivo. Describir las características de una serie de pacientes con infección por C.difficile . Materiales y métodos. Se hizo un estudio descriptivo de una serie de casos de pacientes con infección por C. difficile atendidos en la Fundación Clínica Shaio, entre enero de 2012 y noviembre de 2015. Resultados. Se estudiaron 36 pacientes con una edad promedio de 65 años. Se determinaron los siguientes factores relacionados con la infección por C. difficile: uso previo de antimicrobianos (94,4 %), hospitalización en los últimos tres meses (66,7 %) y uso de inhibidores de la bomba de protones (50 %). Las comorbilidades más comunes fueron la enfermedad renal crónica (41,7 %) y la diabetes mellitus (30,6 %). Los síntomas más frecuentes fueron más de tres deposiciones diarreicas (97,1 %) y dolor abdominal (42,9 %). En cuanto a la gravedad de los casos, 44,4 % se clasificó como leve a moderado, 38,9 % como grave, y 11,1 % como complicado o grave. El método de diagnóstico más utilizado (63,8% de los pacientes) fue la identificación de la toxina mediante reacción en cadena de la polimerasa (PCR). La mortalidad global durante la hospitalización fue de 8 %. Se identificaron cuatro cepas del serotipo NAP1/027 y nueve muestras fueron positivas para la toxina binaria. Conclusión. La infección por C. difficile debe sospecharse en pacientes con deposiciones diarreicas y factores asociados tradicionalmente a esta enfermedad. Se reportó la circulación de cepas hipervirulentas del serotipo NAP1/027 en Colombia, lo cual debe enfrentarse con la vigilancia epidemiológica y el diagnóstico temprano.

Abstract Introduction: Clostridium difficile is the main pathogen related to healthcare-associated diarrhea and it is the cause of 20 to 30% of diarrhea cases caused by antibiotics. In Colombia and Latin America, the knowledge about the epidemiological behavior of this infection is limited. Objective: To describe the characteristics of a series of patients with C. difficile infection. Materials and methods: We performed a descriptive case series study of patients with C. difficile infection hospitalized in the Fundación Clínica Shaio from January, 2012, to November, 2015. Results: We analyzed 36 patients. The average age was 65 years. The risk factors associated with the infection were: previous use of antibiotics (94.4%), prior hospitalization in the last three months (66.7%) and use of proton pump inhibitors (50%). The most common comorbidities were chronic kidney disease (41.7%) and diabetes mellitus (30.6%). The most frequent symptoms were more than three loose stools per day (97.1%) and abdominal pain (42.9%). According to the severity of the disease, 44.4% of cases were classified as mild to moderate, 38.9% as severe, and 11.1% as complicated or severe. The detection of the toxin by PCR (GeneXpert) was the most common diagnostic procedure (63.8%). Global mortality during hospitalization was 8%. We identified four strains with serotype NAP1/027 and nine samples positive for binary toxin. Conclusion: Clostridium difficile infection should be suspected in patients with diarrhea and traditional risk factors associated with this disease. We report the circulation of the hypervirulent strain serotype NAP1/027 in Colombia, which should be countered with epidemiological surveillance and a prompt diagnosis.

Detection of vancomycin-resistant enterococci (VRE) in stool specimens submitted for Clostridium difficile toxin testing

Abstract The aim of this study was to determine the association between Clostridium difficile (C. difficile) and vancomycin-resistant Enterococcus (VRE) and efficacy of screening stools submitted for C. difficile toxin assay for prevalence of VRE. Between April 2012 and February 2014, 158 stool samples submitted for C. difficile toxin to the Marmara University Microbiology Laboratory, were included in the study. Stool samples were analyzed by enzyme immuno assay test; VIDAS (bioMerieux, France) for Toxin A&B. Samples were inoculated on chromID VRE (bioMerieux, France) and incubated 24 h at 37 °C. Manuel tests and API20 STREP (bioMerieux, France) test were used to identify the Enterococci species. After the species identification, vancomycin and teicoplanin MIC's were performed by E test and molecular resistance genes for vanA vs vanB were detected by polymerase chain reaction (PCR). Of the 158 stool samples, 88 were toxin positive. The prevalence of VRE was 17%(n:19) in toxin positives however, 11.4% in toxin negatives(n:70). All VRE isolates were identified as Enterococcus faecium. These results were evaluated according to Fischer's exact chi-square test and p value between VRE colonization and C. difficile toxin positivity was detected 0.047 (p < 0.05). PPV and NPV were 79% and 47% respectively. In our study, the presence of VRE in C. difficile toxin positives is statistically significant compared with toxin negatives (p < 0.05). Screening for VRE is both additional cost and work load for the laboratories. Therefore VRE screening among C. difficile toxin positive samples, will be cost effective for determination of high risk patients in the hospitals especially for developing countries.

Can microcystins affect zooplankton structure community in tropical eutrophic reservoirs? / Microcistinas podem afetar a estrutura da comunidade zooplanctônica em reservatórios eutróficos tropicais?

Abstract The aim of our study was to assess whether cyanotoxins (microcystins) can affect the composition of the zooplankton community, leading to domination of microzooplankton forms (protozoans and rotifers). Temporal variations in concentrations of microcystins and zooplankton biomass were analyzed in three eutrophic reservoirs in the semi-arid northeast region of Brazil. The concentration of microcystins in water proved to be correlated with the cyanobacterial biovolume, indicating the contributions from colonial forms such as Microcystis in the production of cyanotoxins. At the community level, the total biomass of zooplankton was not correlated with the concentration of microcystin (r2 = 0.00; P > 0.001), but in a population-level analysis, the biomass of rotifers and cladocerans showed a weak positive correlation. Cyclopoid copepods, which are considered to be relatively inefficient in ingesting cyanobacteria, were negatively correlated (r2 = – 0.01; P > 0.01) with the concentration of cyanotoxins. Surprisingly, the biomass of calanoid copepods was positively correlated with the microcystin concentration (r2 = 0.44; P > 0.001). The results indicate that allelopathic control mechanisms (negative effects of microcystin on zooplankton biomass) do not seem to substantially affect the composition of mesozooplankton, which showed a constant and high biomass compared to the microzooplankton (rotifers). These results may be important to better understand the trophic interactions between zooplankton and cyanobacteria and the potential effects of allelopathic compounds on zooplankton.

Resumo Com o objetivo de avaliar se as cianotoxinas (microcistinas) podem afetar a composição da comunidade zooplanctônica, levando à dominância de formas microzooplanctônicas (protozoários e rotiferos), as variações nas concentrações de microcistina e a biomassa do zooplâncton foram analisadas em três reservatórios eutróficos na região semi-árida do nordeste brasileiro. A concentração de microcistinas na água esteve correlacionada com o biovolume de cianobactérias, indicando a contribuição de formas coloniais como Microcystis na produção de cianotoxinas. A nível de comunidade, a biomassa total do zooplâncton não apresentou correlacão com a concentração de microcistina (r2 = 0.00; P > 0.001), mas em uma análise a nível de populações, a biomassa de rotíferos e cladóceros apresentou uma fraca correlação positiva. Copépodos Cyclopoida, os quais são considerados relativamente ineficientes na ingestão de cianobactérias, estiveram negativamente correlacionados com a concentração de microcistinas (r2 = - 0.01; P > 0.01). Surpreendentemente, a biomassa de copépodos Calanoida foi positivamente correlacionada com a concentração de cianotoxinas (r2 = 0.44; P > 0.001). Os resultados indicam que mecanismos de controle alelopáticos (efeitos negativos da microcistina sobre o zooplâncton) parecem não afetar substancialmente a composição do mesozooplâncton, que apresentou uma alta e constante biomassa, quando comparada à biomassa do microzooplâncton (rotíferos). Esses resultados podem ser importantes para um melhor entendimento das interações tróficas entre o zooplâncton e cianobactérias, e do efeito potencial de compostos alelopáticos sobre o zooplâncton.

Infección diseminada por Staphylococcus aureus positivo para leucocidina de Panton-Valentine en un niño / Disseminated Panton-Valentine Leukocidin-Positive Staphylococcus aureus infection in a child

La leucocidina de Panton-Valentine (LPV) es una exotoxina producida por muchas cepas de Staphylococcus aureus, y un importante factor de virulencia. Una infección por S. aureus positivo para LPV deriva en infecciones rápidas y graves de partes blandas y neumonía necrosante en adolescentes sanos, y la tasa de mortalidad es elevada. Presentamos el caso de un paciente de 12 años hospitalizado por fiebre, dificultad respiratoria y coxalgia en el que se identificó neumonía necrosante con embolia pulmonar séptica, absceso del psoas, celulitis y osteomielitis. En el hemocultivo del paciente se aisló S. aureus sensible a la meticilina (SASM) positivo para LPV.

Panton-Valentine leukocidin (PVL) is an exotoxin that is produced by many strains of Staphylococcus aureus, and an important virulence factor. A PVL-positive S. aureus infection leads to rapid and severe infections of soft tissue and necrotizing pneumonia in healthy adolescents, and has a high mortality. This case report included a 12-year-old male patient who admitted for fever, respiratory distress and hip pain and was identified with necrotizing pneumonia with septic pulmonary embolism, psoas abscess, cellulitis and osteomyelitis. The PVL positive methicillin-sensitive S. aureus (MSSA) was isolated in the patient blood culture.

Occurrence and toxicity of Microcystis aeruginosa (Cyanobacteria) in the Paraná River, downstream of the Yacyretá dam (Argentina) / Presencia y toxicidad de Microcystis aeruginosa (Cianobacteria) en el río Paraná, aguas abajo de la represa Yacyretá (Argentina)

AbstractCyanobacteria constitute the main toxin producers in inland water ecosystems and have extensive global distribution. The presence of hepatotoxins in aquatic environments is hazardous to human and animal health; even though the presence and identification of hepatotoxic microcystins in rivers and reservoirs of the world have been confirmed by several studies in the last few years. Herein, we studied the abundance and toxicity of Microcystis aeruginosa in the Argentine section of the Paraná River at the beginning of the Middle Paraná (Corrientes Hydrometer), near Corrientes city (27º28´ S - 58º51´ W) and approximately 220 km downstream of the Yacyretá dam (High Paraná). The Paraná River basin, with a drainage area of 3.1 x 106 km2 and 3 965 km in length, is the second largest catchment of South America, after that of the Amazon. The Paraná River is the main source of drinking water supply for the Northeastern Argentine region. Phytoplankton samples were collected and environmental variables were measured in a monthly basis (exceptionally fortnightly), from March 2004 to June 2008. Fifty-eight samples were analyzed for phytoplankton density and biomass. Five samples were used for toxicity testing; the latter were obtained during the cyanobacteria blooms from 2005 to 2008. Phytoplankton counts were performed with an inverted microscope, and biomass was expressed as biovolume. Bioassays with mice and high-performance liquid chromatography (HPLC) analysis were performed to evaluate the presence of cyanotoxins. Phytoplankton mainly consisted of Cryptophyta, Chlorophyta and Bacillariophyta. Microcystis aeruginosa was identified during the warmer months each year (November to March). Density varied between 189 and 25 027 cells/mL (1-10 colonies/mL) and biomass from 0.34 to 44 mm3/L. Taking into account the number of cells, the highest abundance occurred in April 2004 (25 027 cells/mL), coinciding with the largest biovolume (44 mm3/L). All mice subjected to intraperitoneal injections with samples obtained during bloom episodes showed positive results for the presence of hepatotoxins. Three microcystins variants: LR, RR and [D-Leu1] Mcyst-LR were detected by analysis with semi-preparative high-performance liquid chromatography with diode array detector system (HPLC-PDA). This constitutes the first report of microcystins recorded during M. aeruginosa blooms in the Argentine stretch of the Paraná River at the beginning of the Middle Paraná (Corrientes Hydrometer), approximately 220 km downstream of the Yacyretá dam (High Paraná).

ResumenLas Cyanobacterias constituyen el principal productor de toxinas en ecosistemas acuáticos y tienen una amplia distribución mundial. La presencia e identificación de microcistinas hepatotóxicas en ríos y embalses de todo el mundo fue confirmada por diferentes estudios durante los últimos años. La presencia de hepatotoxinas en cuerpos de agua son riesgosas para la salud humana y animal. Se estudió la abundancia y toxicidad de Microcystis aeruginosa (Kütz.) Kütz. en el río Paraná (Argentina), cerca de la ciudad de Corrientes (27°28' S - 58°51' W), aproximadamente a 220 km aguas abajo de la represa Yacyretá. La cuenca del río Paraná, con un área de drenaje de 3.1 x 106 km2 y 3 965 km de longitud, es la segunda mayor cuenca de Sudamérica, después del Amazonas. El río Paraná es la principal fuente de abastecimiento de agua potable para el Nordeste de la República Argentina. Los muestreos se realizaron mensualmente (excepcionalmente fueron quincenales) con medición de variables ambientales, entre Marzo 2004 y Junio 2008. Se tomaron un total de 58 muestras para analizar la densidad y biomasa del fitoplancton; mientras que cinco muestras fueron utilizadas en ensayos de toxicidad, estas últimas fueron obtenidas durante floraciones de cianobacterias entre 2005 y 2008. Los recuentos de fitoplancton fueron realizados con un microscopio invertido y la biomasa fue expresada como biovolumen. Para determinar la presencia de cianotoxinas se utilizaron bioensayos con ratones y análisis con Cromatografia líquida de alta resolución (HPLC). El fitoplancton estuvo representado principalmente por Cryptophyta, Chlorophyta y Bacillariophyta. Cyanobacteria fue dominante durante los meses cálidos de cada año (Noviembre a Marzo), con alta densidad de Microcystis aeruginosa. La densidad de M. aeruginosa varió entre 189 y 25 027 cells/mL (1-10 colonies/mL) y la biomasa entre 0.34 y 44 mm3/L. Teniendo en cuenta el número de células, la mayor abundancia ocurrió en abril 2004 (25 027 cells/ mL), coincidiendo con el gran biovolumen (44 mm3/L). Todos los ratones inyectados intraperitonealmente presentaron síntomas correspondientes a hepatotoxicidad. Tres variantes de microcystinas: LR, RR y [D-Leu1] Mcyst-LR, fueron detectadas por análisis de cromatografía líquida de alta resolución con detector de diodos (HPLC-PDA). Este es el primer trabajo de microcistinas registradas durante las floraciones de M. aeruginosa en el tramo argentino del río Paraná en los inicios del Paraná Medio (Hidrómetro Corrientes), aproximadamente a 220 km aguas abajo de la represa de Yacyretá (Alto Paraná).

Evaluation of a Rapid Membrane Enzyme Immunoassay for the Simultaneous Detection of Glutamate Dehydrogenase and Toxin for the Diagnosis of Clostridium difficile Infection

We evaluated the new C. DIFF QUIK CHEK COMPLETE (CD COMPLETE; TechLab, USA), which is a rapid membrane enzyme immunoassay that uses a combination of glutamate dehydrogenase (GDH) antigen and toxin A and B detection. A total of 608 consecutive loose stool specimens collected from the patients with suspected Clostridium difficile infection (CDI) from August to December 2012 were subjected to the CD COMPLETE and VIDAS Clostridium difficile A & B (VIDAS CDAB; bioMerieux, France). Their performances were compared with a toxigenic culture as a reference. Stool specimens that were culture-negative and CD COMPLETE- or VIDAS CDAB-positive were analyzed by using an enrichment procedure. In comparison to the toxigenic cultures, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were 63.6%, 98.0%, 76.1%, and 96.4%, respectively, for the CD COMPLETE-toxin and 75.5%, 97.4%, 72.5%, and 97.8%, respectively, for the VIDAS CDAB. In comparison to the enriched C. difficile cultures, the sensitivity, specificity, PPV, and NPV for the CD COMPLETE-GDH were 91.0%, 92.4%, 70.5%, and 98.1%, respectively. The CD COMPLETE is a reliable method for the diagnosis of CDI and provides greater sensitivity than toxin enzyme immunoassay alone. Furthermore, the CD COMPLETE-GDH has advantages over direct culture in detecting C. difficile.

Virulence profiles of Shiga toxin-producing Escherichia coli and other potentially diarrheagenic E.coli of bovine origin, in Mendoza, Argentina

This study described a group of strains obtained from a slaughter house in Mendoza, in terms of their pathogenic factors, serotype, antibiotype and molecular profile. Ninety one rectal swabs and one hundred eight plating samples taken from carcasses of healthy cattle intended for meat consumption were analyzed. Both the swab and the plate samples were processed to analyze the samples for the presence of virulence genes by PCR: stx1, stx2, eae and astA. The Stx positive strains were confirmed by citotoxicity assay in Vero cells. The isolates were subsequently investigated for their O:H serotype, antimicrobial susceptibility and molecular profile by Random Amplification of Polymorphic DNA (RAPD). Twelve E.coli strains were identified by their pathogenicity. Nine were from fecal origin and three from carcasses. Three strains carried the stx1 gene, three the stx2 gene, two carried eae and four the astA gene. The detected serotypes were: O172:H-; O150:H8; O91:H21; O178:H19 and O2:H5. The strains showed a similarity around 70% by RAPD. Some of the E.coli strains belonged to serogroups known for certain life-threatening diseases in humans. Their presence in carcasses indicates the high probability of bacterial spread during slaughter and processing.

Posible papel de Bacteroides fragilis enterotoxigénico en la etiología de la vaginitis infecciosa / Possible role of enterotoxigenic Bacteroides fragilis in the etiology of infectious vaginitis

La vaginitis es un trastorno ginecológico frecuente producido por distintas causas, algunas de las cuales permanecen desconocidas. Bacteroides fragilis es el anaerobio más importante en bacteriología clínica. Algunas cepas son enterotoxigénicas y se asocian con síndromes intestinales y extraintestinales. Recientemente han sido aisladas de pacientes con vaginitis. En este trabajo se planteó investigar la posible asociación de B. fragilis enterotoxigénico con la vaginitis infecciosa. Fueron procesadas 265 muestras de exudado vaginal. 202 de mujeres sintomáticas y 63 mujeres sanas. La identificación de los microorganismos se realizó por métodos convencionales. En 31,2% de las pacientes sintomáticas se identificaron: Gardnerella vaginalis, Candida albicans, Mobiluncus, Mycoplasma hominis, Ureaplasma urealyticum y Streptococcus agalactiae. En 27 pacientes sintomáticas y en 5 mujeres sanas se identificó B. fragilis. Estas cepas fueron cultivadas en medio líquido e incubadas durante 48 h a 36° C en anaerobiosis. La toxicidad en los sobrenadantes se ensayó en células HT-29. 18 cepas de B. fragilis aisladas de pacientes sintomáticas fueron enterotoxigénicas, ya que indujeron alteraciones en la monocapa celular y en las células. No se identificó en mujeres sanas (P<0,05). 77,7% de las cepas de B. fragilis enterotoxigénicas no se encontraron asociadas con otros patógenos específicos. Este hecho sugiere que pudiera ser un agente causante de vaginitis, ya que el efecto de la enterotoxina sobre la E-cadherina del epitelio vaginal podría facilitar la invasión y su posible papel patógeno en la vagina. Esta es la primera investigación que asocia a Bacteroides fragilis enterotoxigénico como posible causa de vaginitis infecciosa.

Vaginitis is a common gynecologic disorder. It is due to several causes, some even unknown. Bacteroides fragilis is the most important anaerobe in clinical bacteriology, some strains of this group are notable for being enterotoxigenic and they have been associated with intestinal and extraintestinal syndromes. They have recently been isolated from patients with vaginitis. The purpose of this study was to investigate a possible association of enterotoxigenic B. fragilis with infectious vaginitis. 265 samples of vaginal exudate were processed, 202 from symptomatic patients and 63 healthy women. The identification of the microorganisms was carried out by conventional methods. In 31.2% of symptomatic patients were identified: Gardnerella vaginalis, Mobiluncus, Candida albicans, Mycoplasma hominis, Ureaplasma urealyticum and Streptococcus agalactiae. B. fragilis was identified in 27 symptomatic patients and 5 healthy women. These strains were cultivated in liquid medium and incubated during 48 h at 36°C in anaerobe chambers. Supernatant activity was assayed in HT-29 cells. Eighteen B. fragilis strains isolated from symptomatic patients were enterotoxigenic, because induced alterations in target cell morphology. It was not identified in healthy women (P<0.05). 77.7% of enterotoxigenic B. fragilis strains were not associated with other specific pathogens. This fact suggests that enterotoxigenic B. fragilis could be a cause for vaginitis. The effect of enterotoxin on E-cadherin of vaginal epithelium could facilitate invasion and its possible pathogenic role in the vagina. This is the first report that associates enterotoxigenic Bacteroides fragilis as a possible cause of infectious vaginitis.

Clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile cultivated from stool samples of hospitalized patients

The aim of this study was to fortify the clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile isolated from stool samples of hospitalized patients. This survey included 80 hospitalized patients with diarrhea and positive findings of Clostridium difficile in stool samples, and 100 hospitalized patients with formed stool as a control group. Bacteriological examination of a stool samples was conducted using standard microbiological methods. Stool sample were inoculated directly on nutrient media for bacterial cultivation (blood agar using 5% sheep blood, Endo agar, selective Salmonella Shigella agar, Selenite-F broth, CIN agar and Skirrow's medium), and to selective cycloserine-cefoxitin-fructose agar (CCFA) (Biomedics, Parg qe tehnicologico, Madrid, Spain) for isolation of Clostridium difficile. Clostridium difficile toxin was detected by ELISA-ridascreen Clostridium difficile Toxin A/B (R-Biopharm AG, Germany) and ColorPAC ToxinA test (Becton Dickinson, USA). Examination of stool specimens for the presence of parasites (causing diarrhea) was done using standard methods (conventional microscopy), commercial concentration test Paraprep S Gold kit (Dia Mondial, France) and RIDA®QUICK Cryptosporidium/Giardia Combi test (R-Biopharm AG, Germany). Examination of stool specimens for the presence of fungi (causing diarrhea) was performed by standard methods. All stool samples positive for Clostridium difficile were tested for Rota, Noro, Astro and Adeno viruses by ELISA - ridascreen (R-Biopharm AG, Germany). In this research we isolated 99 Clostridium difficile strains from 116 stool samples of 80 hospitalized patients with diarrhea. The 53 (66.25%) of patients with diarrhea were positive for toxins A and B, one (1.25%) were positive for only toxin B. Non-toxigenic Clostridium difficile isolated from samples of 26 (32.5%) patients. However, other pathogenic microorganisms of intestinal tract cultivated from samples of 16 patients. Examination of cultivated colonies revealed that most of cultivated species belonged to genera of Campylobacter spp., Salmonella spp., and Candida spp.. In control group, toxigenic Clostridium difficile cultivated from stool samples of two patients (2%) and non-toxigenic Clostridium difficile from samples of five patients (5%). This research confirmed clinical importance of toxigenic Clostridium difficile found in liquid stool samples of hospitalized patient, and the possibility of asymptomatic carriage in 2% of patients with formed stool.

Nasal carriage of Panton Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus in healthy preschool children / Colonización nasal de Staphylococcus aureus Meticilino Resistente portador de la Leucocidina Panton-Valentine en preescolares

Objective Determining the prevalence of nasal carriage of S. aureus, both sensitive to methicillin and resistant to it, in preschool children and evaluating the presence of Panton-Valentine leukocidin genes in the isolates. Methods This was a cross-sectional study in which cultures from anterior nares were obtained from healthy preschool children. Isolates were identified as S. aureus based on morphological and biochemical tests. Antibiotic susceptibility profiles were determined by the disk diffusion method. All the isolates were further analyzed by multiplex PCR to determine the presence of mecA and PVL genes; methicillin-resistant isolates were also SCCmec typed by multiplex PCR. Results Overall S. aureus nasal colonization prevalence was 38.5 % and 4.8 % for methicillin-resistant strains. All the methicillin-resistant isolates carried the genes for PVL; two isolates possessed the SCCmec type IV, two were SCCmec type I and one was SCCmec type II. Conclusion This study revealed high PVL-positive, methicillin-resistant S. aureus colonization prevalence in healthy preschool children from Cartagena, which may play a key role in the epidemiology of community-associated infection by methicillin-resistant S. aureus in healthy children from this particular geographical area.

Objetivo Determinar la prevalencia de colonización nasal de S. aureus, tanto sensible como resistente a meticilina, en niños preescolares y evaluar la presencia de los genes de la leucocidina Panton-Valentine en estos aislamientos. Métodos Estudio de corte transversal en el que se realizaron cultivos de flora nasal de niños preescolares. Los aislamientos fueron identificados como S. aureus con base en su morfología y pruebas bioquímicas. La susceptibilidad a antibióticos se determinó por el método de difusión en disco. Todos los aislamientos fueron analizados por PCR múltiple para determinar la presencia de los genes mecA y PVL, y para la tipificación del casete cromosómico SCCmec de los aislamientos resistentes a meticilina. Resultados La colonización nasal por S. aureus fue 38,5 %, y la de cepas meticilino-resistentes fue 4,8 %. Todos los aislamientos SARM portaban los genes para PVL, dos portaban el elemento SCCmec tipo IV, dos fueron tipo I y uno fue tipo II. Conclusión Encontramos una alta prevalencia de colonización por cepas meticilino-resistentes, PVL-positivos en la población estudiada, lo que podría jugar un papel clave en la epidemiología de las infecciones por S.aureus meticilino-resistente en esta área geográfica.

Comparison between the two-step and the three-step algorithms for the detection of toxigenic Clostridium difficile.

Purpose: To evaluate usefulness of applying either the two-step algorithm (Ag-EIAs and CCNA) or the three-step algorithm (all three assays) for better confirmation of toxigenic Clostridium difficile. The antigen enzyme immunoassays (Ag-EIAs) can accurately identify the glutamate dehydrogenase antigen of toxigenic and nontoxigenic Clostridium difficile. Therefore, it is used in combination with a toxin-detecting assay [cell line culture neutralization assay (CCNA), or the enzyme immunoassays for toxins A and B (TOX-A/BII EIA)] to provide specific evidence of Clostridium difficile-associated diarrhoea. Materials and Methods: A total of 151 nonformed stool specimens were tested by Ag-EIAs, TOX-A/BII EIA, and CCNA. All tests were performed according to the manufacturer's instructions and the results of Ag-EIAs and TOX-A/BII EIA were read using a spectrophotometer at a wavelength of 450 nm. Results: A total of 61 (40.7%), 38 (25.3%), and 52 (34.7%) specimens tested positive with Ag-EIA, TOX-A/BII EIA, and CCNA, respectively. Overall, the sensitivity, specificity, negative predictive value, and positive predictive value for Ag-EIA were 94%, 87%, 96.6%, and 80.3%, respectively. Whereas for TOX-A/BII EIA, the sensitivity, specificity, negative predictive value, and positive predictive value were 73.1%, 100%, 87.5%, and 100%, respectively. With the two-step algorithm, all 61 Ag-EIAs-positive cases required 2 days for confirmation. With the three-step algorithm, 37 (60.7%) cases were reported immediately, and the remaining 24 (39.3%) required further testing by CCNA. By applying the two-step algorithm, the workload and cost could be reduced by 28.2% compared with the three-step algorithm. Conclusions: The two-step algorithm is the most practical for accurately detecting toxigenic Clostridium difficile, but it is time-consuming.

The prevalence of enterotoxigenic E. coli isolated from the stools of children aged 0-10 years with diarrhea in mid-anatolia region, Turkey

The stool samples from 245 patients with diarrhea were tested for heat labile toxin (LT) and heat stable toxins (ST) by passive latex agglutination and enzyme immunoassay methods respectively. Twelve (4.9 percent) enterotoxigenic E. Coli ETEC strains were isolated. Five strains (2 percent) expressed ST, and 7 (2.8 percent) expressed LT.

Evolutionary dynamics of Helicobacter pylori cagA and vacA genes in Iran and their association with clinical outcomes

There is a relationship between specific genotypes of Helicobacter pylori cagA and vacA genes and the increased risk of peptic ulcer diseases and gastric cancer. These genes also possess strong patterns of geographical differentiation. The present study aims to determine the patterns of variation of the virulence genes in Iran and their association with clinical status. Sequence fragments for cagAand vacA were obtained from a total of 147 H. pylori isolates from diverse geographical and ethnic sources within Iran. We used phylogenetic methods to determine the patterns of allelic diversity, and the relationship between evolutionary lineages and clinical status. Phylogenetic analyses of Iranian cagA gene disclosed four lineages, whereas the vacA gene had two distinct lineages. The cagA lineage II showed extensive genetic diversity compared with lineage I. cagA lineages III and IV disclosed mixed ancestries with recombinant nucleotides that originated from lineages I and H Iranian strains with vac A lineage II genotype were significantly cagA+ [> 90%, p = 0.0] and correlated with a higher rate of peptic ulcers in infected individuals [p =0.003]. Most strains in the cagA lineage I showed a vacA lineage II genotype [p = 0.003] and significantly correlated with an increased risk of peptic ulcers in infected individuals [p = 0.022]. Strains with cagA lineage III genotype significantly correlated with gastritis [p = 0.0]. The increased level of allelic diversity in the virulence genes shows strong evolutionary dynamics, resulting in the emergence of new clonal genealogies of the cagA gene within Iran. Particular lineages of the Iranian cagA and vac A genes correlate with peptic ulcer diseases

Investigação da biossíntese de toxinas produzidas por cepas de cianobactérias / Investigation on the cyanobacterial strains toxins biossinthesys

A demanda crescente de água doce de boa qualidade são problemas atuais e mundiais, além do descaso com os dejetos lançados nos ambientes aquáticos que comprometem a qualidade dos recursos hídricos. Um dos parâmetros que atesta a potabilidade da água é a presença de cianobactérias e cianotoxinas. Cianobactérias são microrganismos procariontes aeróbicos fotoautróficos que sintetizam as cianotoxinas. Estes compostos podem ser classificados de acordo com seus mecanismos de ação em hepatotóxicos, neurotóxicos e dermatotóxicos. Por sua diversidade, representam diferentes riscos não só ao ecossistema e a outros organismos dos ambientes aquáticos, como também aos seres humanos. Esse projeto visou o isolamento e cultivo de cepas de cianobactérias produtoras de toxinas para a investigação da biossíntese desses compostos. Com este intuito, foram realizadas coletas de água em três reservatórios no estado de São Paulo e um no Paraná. Cepas de cianobactérais foram isoladas, identificadas e analisadas quanto à produção de toxinas. Uma cepa de Microcystis aeruginosa (LTPNA 02) produtora de microcistinas (MC-LR, MC-RR, MC-YR, MC-LF, MC-LW e desm-MC-LR e desm- MC-RR) foi escolhida para ser estudada frente diferentes condições de cultivo e ter o seu crescimento, produção de toxinas e expressão gênica estudados. Foram utilizados os meios de cultura já referidos na literatura: ASM-1 (N:P=1, 10 e 20), MLA (N:P=10), Bold 3N (N:P=16) e BG-11 (N:P=10 e 100). Para acompanhar o crescimento, dois métodos foram utilizados: contagem de células e espectrofotometria. As toxinas foram quantificadas por LC-MS - QTrap. A análise da expressão gênica foi realizada por reação de PCR em tempo real pelo método de quantificação relativa ΔΔCt. Foi observada diferença no crescimento da cepa estudada nos diferentes meios de cultivo empregados. A contagem das células permitiu a identificação das fases logarítmica e total de crescimento. Durante a fase logarítmica, três experimentos demonstraram...

There is a great concern these days about potable and good quality water due to the increase of the population needs and also to the arising problems with contamination caused by anthropogenic sources. The presence of cyanobacteria and cyanotoxins are some parameters that attest water potability. Cyanobacteria are prokaryotic aerobic photoautotrophic microorganisms that may synthesize cyanotoxins. These compounds can be classified as hepatotoxic, neurotoxic and dermatotoxic according to their action mechanisms. Because of their diversity, they may represent different risks, not only to their ecosystem and other aquatic living organisms, but also to human beings. The aim of this project was the isolation and cultivation of cyanotoxin-producing cyanobacteria for further investigation on the biosynthesis of these compounds. Water samples from three different reservoirs in São Paulo state and one in Paraná state were collected in order to isolate cyanobacteria strains and accomplish their identification and to evaluate the toxin production. The Microcystis aeruginosa (LTPNA 02) microcystin producer strain (MCLR, MC-RR, MC-YR, MC-LF, MC-LW, desm-MC-LR and desm-MC-RR) was chosen to be grown in different cultivation conditions and later analyzed for its growth rate, toxin production and gene expression. All culture media used in this research were chosen according to the literature: ASM-1 (N:P=1, 10 and 20), MLA (N:P=10), Bold 3N (N:P=16) and BG-11 (N:P=10 and 100). To evaluate growth rate, two techniques were used: cell counting and absorbance determination in two different wavelengths (680 nm and 750 nm). Toxins were quantified by LC-MS in a hybrid triple-quadrupole instrument (Qtrap). Gene expression was assessed by real time PCR, using the ΔΔCt relative quantification method. Cell counting allowed total growth and logarithmic phase identification. During the last, three experiments showed statistical difference from control group (p<0,05). Four experiments...

Epidemiology and Clinical Characteristics of Clostridium difficile Infection in a Korean Tertiary Hospital

In order to investigate the incidence, clinical and microbiologic characteristics of Clostridium difficile infection (CDI) in Korea, a prospective observational study was performed. From September 2008 through January 2010, all patients whose stool was tested for toxin assay A&B and/or C. difficile culture were studied for clinical characteristics. Toxin types of the isolates from stool were tested. The mean incidence of CDI per 100,000 patient-days was 71.6 by month (range, 52.5-114.0), and the ratio of CDI to antibiotic-associated diarrhea was 0.23. Among 200 CDI patients, 37.5% (75/200) was severe CDI based on severity score. Clinical outcome of 189 CDI was as followed; 25.9% (49/189) improved without treatment, 84.3% (118/140) achieved clinical cure and attributed mortality was 0.7% (1/140) with the treatment. Recurrence rate was 21.4% (30/140) and cure without recurrence was 66.4% (93/140). The most common type of toxin was toxin A-positive/toxin B-positive strain (77.5%), toxin A-negative/toxin B-positive strains or binary toxin-producing strains comprised 15.4% or 7.1%, respectively. In conclusion, the incidence of CDI in Korea is a little higher than other reports during the non-epidemic setting. We expect that the change of epidemiology and clinical severity in CDI can be evaluated based on these results.

Virulence factors and genetic variability of uropathogenic Escherichia coli isolated from dogs and cats in Italy

In this study, the association between virulence genotypes and phylogenetic groups among Escherichia (E.) coli isolates obtained from pet dogs and cats with cystitis was detected, and fingerprinting methods were used to explore the relationship among strains. Forty uropathogenic E. coli (UPEC) isolated from dogs (n = 30) and cats (n = 10) in Italy were analysed by polymerase chain reaction (PCR) for the presence of virulence factors and their classification into phylogenetic groups. The same strains were characterized by repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR techniques. We found a high number of virulence factors such as fimbriae A, S fimbriae (sfa) and cytotoxic necrotizing factor 1 (cnf1) significantly associated with phylogenetic group B2. We demonstrated a high correlation between alpha-hemolysin A and pyelonephritis C, sfa, and cnf1 operons, confirming the presence of pathogenicity islands in these strains. In addition, UPEC belonging to group B2 harboured a greater number of virulence factors than strains from phylogenetic groups A, B1, and D. REP- and ERIC-PCR grouped the UPEC isolates into two major clusters, the former grouping E. coli strains belonging to phylogenetic group B2 and D, the latter grouping those belonging to groups A and B1. Given the significant genetic variability among the UPEC strains found in our study, it can be hypothesized that no specific genotype is responsible for cystitis in cats or dogs.