Molecular quantification and differentiation of Candida species in biological specimens of patients with liver cirrhosis

Patients with liver cirrhosis are susceptible to fungal infections. Due to low sensitivity of culture-based methods, we applied a real-time PCR assay targeting the 18S rRNA gene in combination with direct sequencing and terminal-restriction fragment length polymorphism (T-RFLP) in order to establish a novel tool to detect fungal DNA and to quantify and differentiate Candida DNA, also in polyfungal specimens. In total, 281 samples (blood n=135, ascites n=92, duodenal fluid n=54) from 135 patients with liver cirrhosis and 52 samples (blood n=26, duodenal fluid n=26) from 26 control patients were collected prospectively. Candida DNA was quantified in all samples. Standard microbiological culture was performed for comparison. Blood and ascites samples, irrespective of the patient cohort, showed a method-independent low fungal detection rate of approximately 1%, and the Candida DNA content level did not exceed 3.0x101 copies ml-1 in any sample. In contrast, in duodenal fluid of patients with liver cirrhosis high fungal detection rates were discovered by using both PCR- and culture-based techniques (81.5% vs. 66.7%; p=0.123) and the median level of Candida DNA was 3.8x105 copies ml-1 (2.3x102-6.3x109). In cirrhosis and controls, fungal positive culture results were confirmed by PCR in 96% and an additional amount of 44% of culture negative duodenal samples were PCR positive. Using T-RFLP analysis in duodenal samples, overall 85% of results from microbial culture were confirmed and in 75% of culture-negative but PCR-positive samples additional Candida species could be identified. In conclusion, PCR-based methods and subsequent differentiation of Candida DNA might offer a quick approach to identifying Candida species without prior cultivation

Patient characteristics at baseline.

<p>Patient characteristics at baseline.</p

Receiver operating characteristic curve (ROC) of 18S rRNA gene based quantitative PCR from biological samples of patients with liver cirrhosis and controls.

<p>Receiver operating characteristic curve (ROC) of 18S rRNA gene based quantitative PCR from biological samples of patients with liver cirrhosis and controls.</p

Fungal pathogen and <i>Candida</i> DNA detection rates from microbial culture, real-time PCR analysis, and its statistical significance.

<p>Fungal pathogen and <i>Candida</i> DNA detection rates from microbial culture, real-time PCR analysis, and its statistical significance.</p

Fungal species analysis using either the T-RFLP or conventional culture methods on duodenal fluid samples from patients with liver cirrhosis and controls (n = 56).

<p>Each bar represents a single species. A star indicates that a fungal species could not be confirmed by the alternative method. The x-axis presents single duodenal samples so that multiple culture or T-RFLP-identified pathogens per sample are stacked in columns. Samples with culture-positive and PCR/T-RFLP-positive samples are compared in A (n = 40). Culture-negative but PCR/T-RFLP-positive samples are displayed in B (n = 9) and culture-positive and PCR/T-RFLP-negative samples are presented in C (n = 2). The duodenal samples that were culture-positive and PCR-positive but T-RFLP-negative are represented in area D (n = 5). Additional duodenal samples that were culture negative but PCR-positive with DNA not being sufficient for T-RFLP analysis (n = 3) have been left out in analysis.</p

Detection of fungal species in blood and duodenal fluid of patients with liver cirrhosis and in control patients by using microbial culture techniques.

<p>The number of each sample group equals 100%. Stacked bars without percentage value correspond to 1.6%.</p

Aeromonas species isolated from PINTADO fish (Pseudoplatystoma sp): virulence factors and drug susceptibility

Aeromonas has been described as an emergent foodborne pathogen of increasing importance. In this study, we report that 48% of 50 Pintado fish samples collected at the retail market of São Paulo city were positive for Aeromonas sp, as detected by the direct plating method. When the presence/absence method was used, the positivity was 42%. A. caviae was the most frequent species, followed by A. hydrophila and A. sobria. Production of cytotoxic enterotoxin, observed in suckling mouse assay, was detected in 67% of A. sobria strains, in 60% of A. hydrophila strains and in 40% of A. caviae strains. In vitro tests, performed with HEp-2 cells, showed that 88% of A. hydrophila, 27% of A. sobria and 13% of A. caviae strains were positive for this toxin. The in vivo production of cytotonic enterotoxin, tested after heating the filtrates at 56ºC for 20 minutes, was detected in 17% of A. sobria, in 10% of A. caviae and in none of A. hydrophila strains in vivo. All analyzed strains did not alter HEp-2 cells. 20% and 16% of A. sobria and A. caviae isolates, respectively, presented capacity to adhere to HEp-2 cells. In counterpart, invasion of HEp-2 cells was not observed in any isolate. The Aeromonas isolates were sensitive to the majority of the antimicrobiol agents tested.<br>Bactérias do gênero Aeromonas têm sido descritas como patógenos emergentes de importância crescente em alimentos. Neste estudo, relatamos que 48% das amostras de peixe "Pintado" coletado no comércio de São Paulo, foram positivas para Aeromonas sp quando isoladas pelo método de plaqueamento direto. Quando o método Presença/Ausência foi utilizado, a porcentagem de positividade foi de 42%. A. caviae foi a espécie mais freqüente, seguida por A. hydrophila e A. sobria. Produção de enterotoxina citotóxica, determinada em camundongos recém-nascidos, foi observada em 67% das cepas de A. sobria, em 60% das de A. hydrophila e em 40% das de A. caviae. No teste in vitro em células HEp- 88% das cepas de A. hydrophila, 27% das cepas de A. sobria e 13% das cepas de A. caviae revelaram-se positivas. Com relação a produção de enterotoxina citotônica, testada após o aquecimento do sobrenadante a 56ºC por 20 minutos, 17% das cepas de A. sobria, 10% das de A. caviae e nenhuma das de A. hydrophila foram positivas in vivo e para todas as cepas analisadas, os testes foram negativos em cultura de célula HEp-2. Quanto a capacidade de adesão, 20% das 5 cepas de A. sobria e 16% das 20 cepas de A. caviae aderiram a células HEp-2. A capacidade de invasão em células HEp-2 não foi detectada em nenhuma das cepas testadas. As cepas isoladas foram sensíveis a maior parte dos antimicrobianos testados