Carga parasitaria determinada por PCR en tiempo real en madres serorreactivas contra trypanosoma cruzi / Parasite load determined by real time PCR in seroreactive mothers against trypanosoma cruzi

INTRODUCCIÓN Una vez que se ha logrado el control vectorial y transfusional, la transmisión congénita por Trypanosoma cruzi (T. cruzi) es la vía y la fuente principal de nuevos casos de enfermedad de Chagas en América Latina y en países no endémicos. OBJETIVOS Evaluar la parasitemia de las madres serorreactivas como variable predictora de transmisión de la infección congénita en la provincia de Santa Fe. MÉTODOS Se seleccionó a madres serorreactivas de diferentes centros y hospitales de las ciudades de Rosario y Santa Fe, a quienes se les realizó cuantificación de la parasitemia por PCR en tiempo real (qPCR) en diferentes trimestres, al fin del embarazo (FE) y en tiempos posteriores al parto. Los datos se analizaron por tests no paramétricos, y se consideró significativo un p<0,05. Resultados Se seleccionó para el análisis a 67 de 75 madres. Un total de 5 niños fueron diagnosticados con infección congénita por T. cruzi por micrométodo y/o PCR. La qPCR fue más elevada cuando las madres trasmitieron la infección congénita a sus hijos al FE o después del parto (test de Kruskal-Wallis, p=0,0040). La media ± DS de los valores de qPCR fue de 130±147 (IC 95% 52,1-313), 10,7±13,6 (IC95% 3,94-17,5) y 5,73±12,7 (IC95%: 1,61-13,1) eq. par/mL en las madres que transmitieron la infección congénita frente a las que no la trasmitieron en el tercer trimestre de gestación, respectivamente. DISCUSIÓN Las madres transmisoras de la ciudad de Santa Fe provenientes de diferentes lugares del norte de la provincia (un área de endemicidad controlada), evaluadas al FE y hasta dos meses después del nacimiento tuvieron una parasitemia significativamente más elevada que en etapas previas de la gestación de madres no transmisoras, lo que indica que la parasitemia parece ser determinante de la transmisión vertical.

Trypanosoma cruzi discrete typing units in Chagas disease patients from endemic and non-endemic regions of Argentina.

Genetic diversity of Trypanosoma cruzi may play a role in pathogenesis of Chagas disease forms. Natural populations are classified into 6 Discrete Typing Units (DTUs) Tc I-VI with taxonomical status. This study aimed to identify T. cruzi DTUs in bloodstream and tissue samples of Argentinean patients with Chagas disease. PCR-based strategies allowed DTU identification in 256 clinical samples from 239 Argentinean patients. Tc V prevailed in blood from both asymptomatic and symptomatic cases and Tc I was more frequent in bloodstream, cardiac tissues and chagoma samples from immunosuppressed patients. Tc II and VI were identified in a minority of cases, while Tc III and Tc IV were not detected in the studied population. Interestingly, Tc I and Tc II/VI sequences were amplified from the same skin biopsy slice from a kidney transplant patient suffering Chagas disease reactivation. Further data also revealed the occurrence of mixed DTU populations in the human chronic infection. In conclusion, our findings provide evidence of the complexity of the dynamics of T. cruzi diversity in the natural history of human Chagas disease and allege the pathogenic role of DTUs I, II, V and VI in the studied population.

[Comparison of diagnostic methods for Clostridium difficile-associated diarrhea]. / Comparación de métodos diagnósticos de diarreas asociadas a Clostridium difficile.

In order to compare different methods for the diagnosis of Clostridium difficile-associated diarrhea, fecal filtrates from patients presenting symptoms compatible with this condition, were analyzed. Biological activity on Vero cells (biological assay), dot blot with antibodies anti-TcdA and anti-TcdB, and a PCR assay for the tcdB gene, were evaluated. Titles of biological assays were > or =64 for 44 out of 177 samples. Nineteen samples were positive in both biological and PCR assays. The analysis by dot blot using anti-TcdA and anti-TcdB antibodies showed that 46 samples out of 149 were positive for both toxins whereas 12 samples were only positive for TcdB, and 5 samples only positive for TcdA. Discrepancies in the different methods could be related to truncated genes, low number of microorganisms in the samples and toxin degradation. The results herein presented show the need for developing diagnostic approaches compatible with the complex epidemiological situation of this clinically relevant intestinal pathogen.

Comparación de métodos diagnósticos de diarreas asociadas a Clostridium difficile / Comparison of diagnostic methods for Clostridium difficile-associated diarrhea

Para comparar diferentes métodos de diagnóstico de diarreas asociadas a Clostridium difficile desarrollados en el marco de un estudio colaborativo, se analizaron filtrados de materia fecal de pacientes con sintomatología compatible con esta patología. Se evaluó la actividad biológica sobre células Vero (ensayo biológico), la reactividad frente a anticuerpos anti-TcdA y anti-TcdB (dot blot) y la presencia de secuencias del gen tcdB por PCR. De 177 muestras analizadas por el ensayo biológico, 44 tuvieron títulos mayores o iguales que 64. Diecinueve muestras fueron a la vez positivas en el ensayo biológico y en el análisis por PCR. Se analizaron 149 muestras por dot blot utilizando anticuerpos anti-TcdA y anti-TcdB; 46 muestras resultaron positivas para ambas toxinas, 12 muestras fueron positivas sólo para TcdB y 5 muestras sólo para TcdA. Las divergencias entre los diferentes métodos podrían estar relacionadas con la presencia de genes truncados, con un bajo número de microorganismos en las muestras analizadas o con la degradación de las toxinas. Los resultados presentados demuestran la necesidad de implementar alternativas diagnósticas que se adapten a la compleja realidad epidemiológica de este importante patógeno intestinal.

In order to compare different methods for the diagnosis of Clostridium difficile-associated diarrhea, fecal filtrates from patients presenting symptoms compatible with this condition, were analyzed. Biological activity on Vero cells (biological assay), dot blot with antibodies anti-TcdA and anti-TcdB, and a PCR assay for the tcdB gene, were evaluated. Titles of biological assays were ≥ 64 for 44 out of 177 samples. Nineteen samples were positive in both biological and PCR assays. The analysis by dot blot using anti-TcdA and anti-TcdB antibodies showed that 46 samples out of 149 were positive for both toxins whereas 12 samples were only positive for TcdB, and 5 samples only positive for TcdA. Discrepancies in the different methods could be related to truncated genes, low number of microorganisms in the samples and toxin degradation. The results herein presented show the need for developing diagnostic approaches compatible with the complex epidemiological situation of this clinically relevant intestinal pathogen.

Phosphorylated PmrA interacts with the promoter region of ugd in Salmonella enterica serovar typhimurium.

The Salmonella PmrA-PmrB system controls the expression of genes necessary for polymyxin B resistance. Four loci were previously identified as part of the regulon, and interaction of PmrA with the promoter region of three of them was observed. Here we characterized the interaction of PmrA with the promoter region of ugd, previously suggested to be regulated indirectly by PmrA. Our results indicate that PmrA controls the expression of ugd by interacting with a specific sequence in the promoter region of this gene.