[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.

Phenotypic and genotypic study of Streptococcus agalactiae in vagina of pregnant women in Argentina.

Streptococcus agalactiae (GBS) is a leading cause of serious neonatal infection. In this study we determine the prevalence, serotype distribution and genomic diversity of GBS in vagina of pregnant women. Vaginal swabs of 531 pregnant women were cultured on Columbia Agar Base Blood, GBS Agar Base and Todd Hewitt Broth. GBS were characterized by group and type-specific agglutination. Genomic polymorphism was studied by random amplification of DNA (RAPD). Seventeen patients (3.2%) were positive for GBS, resulting serotype III the most frequent. RAPD detected 16 different RAPD profiles from 21 GBS studied, revealing a good discriminatory power. In this sense, this method showed different genotype from GBS serotype III recovered from successive samples of two patients, suggesting reinfection. In conclusion, the combination of RAPD and serotyping appear promising for epidemiological studies. Finally, findings of reinfection after therapy during pregnancy, led us to suggest performing prenatal GBS screening and intrapartum prophylaxis in order to reduce neonatal risk.

Phenotypic and genotypic study of Streptococcus agalactiae in vagina of pregnant women in Argentina.

Streptococcus agalactiae (GBS) is a leading cause of serious neonatal infection. In this study we determine the prevalence, serotype distribution and genomic diversity of GBS in vagina of pregnant women. Vaginal swabs of 531 pregnant women were cultured on Columbia Agar Base Blood, GBS Agar Base and Todd Hewitt Broth. GBS were characterized by group and type-specific agglutination. Genomic polymorphism was studied by random amplification of DNA (RAPD). Seventeen patients (3.2

) were positive for GBS, resulting serotype III the most frequent. RAPD detected 16 different RAPD profiles from 21 GBS studied, revealing a good discriminatory power. In this sense, this method showed different genotype from GBS serotype III recovered from successive samples of two patients, suggesting reinfection. In conclusion, the combination of RAPD and serotyping appear promising for epidemiological studies. Finally, findings of reinfection after therapy during pregnancy, led us to suggest performing prenatal GBS screening and intrapartum prophylaxis in order to reduce neonatal risk.

Genomic diversity among Streptococcus agalactiae isolates detected by a degenerate oligonucleotide-primed amplification assay.

A random-amplified polymorphic DNA assay using partially degenerate oligonucleotides as primers was used for the characterization of 78 epidemiologically related and unrelated clinical isolates of Streptococcus agalactiae belonging to different serotypes. Thirty distinct amplification profiles were obtained among 52 unrelated S. agalactiae isolates assigned to nine groups by serotyping (including 3 nontypeable strains), uncovering the extent of genomic heterogeneity existent within serotypes. This method was particularly useful in providing evidence for or against vertical transmission of a given clone of this microorganism, as well as for relapsing or reinfection in related cases, and suggested clonal relatedness between unrelated S. agalactiae isolates associated with some invasive infections. Thus, this simple methodology represents a suitable tool for the epidemiologic study of S. agalactiae infections.

Characterization of clinical isolates of Streptococcus agalactiae by random amplified polymorphic DNA using degenerate oligonucleotides.

Epidemiological studies of Streptococcus agalactiae strains have been limited by the lack of sensitive and discriminatory methods for comparing clinical isolates. Serotyping, albeit a widely used methodology, has been shown to possess low capability to distinguish between epidemiologically related and unrelated isolates. We have employed here a random amplification of polymorphic DNA (RAPD) assay, using degenerate oligonucleotides as primers, to characterize S. agalactiae isolates from related or unrelated clinical samples. Epidemiologically-related isolates (mother-infant pairs) showed identical profiles by this methodology. On the contrary, 12 epidemiologically-unrelated isolates (classified into 5 different serotypes) resulted in 11 distinct RAPD patterns. This suggests that the proposed modified RAPD assay provides a highly discriminatory tool for the analysis of genomic diversity among isolates from pathogenic organisms.

Characterization of clinical isolates of Streptococcus agalactiae by random amplified polymorphic DNA using degenerate oligonucleotides.

Epidemiological studies of Streptococcus agalactiae strains have been limited by the lack of sensitive and discriminatory methods for comparing clinical isolates. Serotyping, albeit a widely used methodology, has been shown to possess low capability to distinguish between epidemiologically related and unrelated isolates. We have employed here a random amplification of polymorphic DNA (RAPD) assay, using degenerate oligonucleotides as primers, to characterize S. agalactiae isolates from related or unrelated clinical samples. Epidemiologically-related isolates (mother-infant pairs) showed identical profiles by this methodology. On the contrary, 12 epidemiologically-unrelated isolates (classified into 5 different serotypes) resulted in 11 distinct RAPD patterns. This suggests that the proposed modified RAPD assay provides a highly discriminatory tool for the analysis of genomic diversity among isolates from pathogenic organisms.