Breakthrough Clostridium difficile Infection in Cirrhotic Patients Receiving Rifaximin.

Background: Patients with cirrhosis are at high risk of Clostridium difficile infection (CDI). Rifaximin is commonly used in cirrhotic patients as prophylaxis for hepatic encephalopathy (HE). Several studies have demonstrated the efficacy of rifaximin in the treatment of CDI; however, resistance to rifaximin has also been reported. Few studies have assessed the risk of developing CDI in cirrhotic patients receiving rifaximin. Our objective was to assess the incidence and characteristics of CDI in patients with cirrhosis, especially in those who received rifaximin. Methods: We assessed the incidence and clinical characteristics of CDI in cirrhotic patients over a 6-year period in our hospital. Medical charts were retrospectively reviewed. Ribotyping and antimicrobial susceptibility testing of all strains against rifaximin were performed. Results: A total of 388 cirrhotic patients were included, of whom 127 patients had at least 1 episode of diarrhea in which a sample was sent to the laboratory. CDI was detected in 46 patients. Fourteen patients (30.4%) were receiving rifaximin as prophylaxis for HE. The main ribotypes detected were 001 (30.4%), followed by 014 (19.6%). Resistance to rifaximin was 34.1% overall, and 84.6% in patients who had received rifaximin. Multivariate analysis showed that rifamycin therapy and ribotype 001 were significant risk factors for having a rifaximin-resistant C. difficile strain. Conclusions: A high percentage of CDI cases were detected in cirrhotic patients receiving rifaximin, mostly owing to selection of rifaximin-resistant C. difficile strains. Clinicians should be aware of the risk of CDI in cirrhotic patients, even in those receiving rifaximin.

Clinical Significance of Clostridium difficile in Children Less Than 2 Years Old: A Case-Control Study.

BACKGROUND: The significance of Clostridium difficile (CD) in the stools of children 2 years old or younger remains unclear. The aim of this study was to investigate risk factors and clinical evolution of diarrheic children ≤2 years old with or without CD in their stools. METHODS: From January 1, 2012 to December 31, 2013, all diarrheic stool samples received in our laboratory were screened for CD. We randomly selected diarrheic children ≤2 years old (n = 100) with an isolation of toxigenic CD in the stools and compared them with diarrheic children (n = 100) without isolation of CD. RESULTS: Cases and controls were appropriately matched for age and sex. We found no significant differences between children with or without CD. Of the CD cases, we compared the patients receiving treatment with metronidazole (19%) versus those that were not prescribed treatment (81%), and found that patients in the first group had used more gastric acid suppressants (P = 0.02), had surgery in the last month (P = 0.03) and also presented with more days with diarrhea (P = 0.03). All the patients, including CD cases, independently of the administration of metronidazole, were cured of the diarrheic episode. Polymerase chain reaction-ribotyping performed in all CD cases showed that the most prevalent ribotype was 014 (25%). CONCLUSIONS: Our study reinforces the nonsignificance of CD in neonates and infants younger than 2 years old. Informing clinicians of CD isolates in this population promotes the use of antibiotics against CD, without evidence of a different outcome than those not receiving treatment.

A new type of toxin A-negative, toxin B-positive Clostridium difficile strain lacking a complete tcdA gene.

Toxins A and B are the main virulence factors of Clostridium difficile and are the targets for molecular diagnostic tests. Here, we describe a new toxin A-negative, toxin B-positive, binary toxin CDT (Clostridium difficile transferase)-negative (A(-) B(+) CDT(-)) toxinotype (XXXII) characterized by a variant type of pathogenicity locus (PaLoc) without tcdA and with atypical organization of the PaLoc integration site.

Clostridium difficile isolates with high linezolid MICs harbor the multiresistance gene cfr.

We studied the molecular mechanisms of linezolid resistance in 9 isolates of toxigenic Clostridium difficile with high linezolid MICs. The activity of linezolid was determined against 891 clinical isolates of toxigenic C. difficile. The MIC50 and MIC90 of linezolid were 0.75 µg/ml and 1.5 µg/ml, respectively. Nine strains (1%) showed high linezolid MICs (6 µg/ml to 16 µg/ml) and also were resistant to clindamycin, erythromycin, and chloramphenicol. These strains were selected for molecular studies: sequencing of domain V of the 23 rRNA gene, detection of the cfr methyltransferase gene, and sequencing of the ribosomal protein genes rplC and rplD. Molecular relatedness between strains was assessed using PCR ribotyping and MLVA (multilocus variable-number tandem-repeat analysis) typing. The strains belonged to ribotypes 001 (2/9), 017 (6/9), and 078 (1/9). MLVA showed that strains of ribotype 001 and 017 belonged to the same clonal complex in each ribotype. We did not detect mutations in the 23S rRNA gene. The cfr gene was detected in 7 of 9 strains. Sequencing of cfr amplicons revealed a similarity of 100% to a fragment of transposon Tn6218 of C. difficile, which was annotated as a putative chloramphenicol/florfenicol resistance protein. We were unable to detect mechanisms of resistance to linezolid in the 2 strains belonging to ribotype 001. While the relevance of our results lies in the detection of the cfr gene as a possible mechanism of resistance to linezolid in C. difficile, our findings should be assessed by further investigations to characterize these possible cfr genes and their contribution to linezolid resistance.

First case of autochthonous Clostridium difficile PCR ribotype 027 detected in Spain / Primer caso autóctono de Clostridium difficile del ribotipo 027 detectado en España

INTRODUCTION: Clostridium difficile ribotype 027 (Cd027) has caused outbreaks in the United States, Canada, and Europe since 2001. In Spain, the importance of Cd027 is still unknown. In 2007, we began active surveillance of Cd027 to determine its incidence in our hospital. Methods From January 2007 to April 2012, isolates of C. difficile by multiplex PCR were studied to detect toxin genes. Binary toxin-positive isolates were characterized using PCR-ribotyping. Cd027 were further characterized by toxino-typing, sequencing of tcdC gene, and MLVA (multilocus-variable-number-tandem-repeat-analysis).Results Only 8 strains were Cd027 from 3666 isolates of C. difficile analyzed during the study period. These strains were isolated from 4 patients: a Spanish patient previously hospitalized in the UK, a pregnant laboratory technician, a British tourist, and a Spanish patient without epidemiological antecedents for acquiring Cd027. MLVA typing of Cd027 isolates revealed 4 different patterns. The first patient had 2 episodes of diarrhea caused by different Cd027. The strains from the first episode of patient 1 and the strain from patient 2 were grouped in the same clonal cluster (these cases were previously published as laboratory transmission), while strains from patients 3 and 4 were genetically unrelated to each other, and to the strains from patients 1 and 2.ConclusionWe report the first finding of an autochthonous case of non-severe Cd027 infection. Our results indicate that Cd027 diarrhea is uncommon in our area, and it appears mainly as imported cases. MLVA typing enables us to distinguish different genotypes among our Cd027 isolates

INTRODUCCIÓN: Clostridium difficile del ribotipo 027 (Cd027) ha causado importantes brotes en EE. UU, Canadá y Europa desde 2001. Actualmente su importancia en España es poco conocida. En 2007, nuestro grupo inició la búsqueda de Cd027 para determinar su incidencia en nuestro hospital. MÉTODOS: Desde enero de 2007 hasta abril de 2012 se estudiaron todos los aislados de C. difficile mediante PCR multiplex de los genes de las toxinas. Las cepas toxina binaria positivas se caracterizaron por PCR-ribotipado. Las cepas de Cd027 encontradas se genotiparon por toxinotipo, secuenciación del gen tcdC y MLVA. RESULTADOS: Durante el periodo de estudio se analizaron 3.666 cepas de C. difficile de las que solo 8 fueron Cd027. Estas cepas se aislaron de 4 pacientes: una paciente española previamente hospitalizada en el Reino Unido, una técnico de nuestro laboratorio, una turista británica y un paciente español sin antecedentes de riesgo para haber adquirido Cd027. Mediante MLVA obtuvimos 4 patrones de tipado diferentes. La primera paciente tuvo 2 episodios de diarrea causados por cepas diferentes de Cd027. Una de estas cepas fue la misma que la de nuestra técnico de laboratorio (este caso está publicado como una transmisión de laboratorio). Las cepas de los pacientes 3 y 4 tuvieron MLVA únicos. CONCLUSIÓN: En este trabajo describimos el primer autóctono de diarrea causada por Cd027. Nuestros resultados indican que es infrecuente en nuestro medio y que aparece principalmente como casos importados. El tipado por MLVA nos ha permitido diferenciar genotipos diferentes entre los aislados de Cd027 de nuestro hospital

First case of autochthonous Clostridium difficile PCR ribotype 027 detected in Spain.

INTRODUCTION: Clostridium difficile ribotype 027 (Cd027) has caused outbreaks in the United States, Canada, and Europe since 2001. In Spain, the importance of Cd027 is still unknown. In 2007, we began active surveillance of Cd027 to determine its incidence in our hospital. METHODS: From January 2007 to April 2012, isolates of C. difficile by multiplex PCR were studied to detect toxin genes. Binary toxin-positive isolates were characterized using PCR-ribotyping. Cd027 were further characterized by toxino-typing, sequencing of tcdC gene, and MLVA (multilocus-variable-number-tandem-repeat-analysis). RESULTS: Only 8 strains were Cd027 from 3666 isolates of C. difficile analyzed during the study period. These strains were isolated from 4 patients: a Spanish patient previously hospitalized in the UK, a pregnant laboratory technician, a British tourist, and a Spanish patient without epidemiological antecedents for acquiring Cd027. MLVA typing of Cd027 isolates revealed 4 different patterns. The first patient had 2 episodes of diarrhea caused by different Cd027. The strains from the first episode of patient 1 and the strain from patient 2 were grouped in the same clonal cluster (these cases were previously published as laboratory transmission), while strains from patients 3 and 4 were genetically unrelated to each other, and to the strains from patients 1 and 2. CONCLUSION: We report the first finding of an autochthonous case of non-severe Cd027 infection. Our results indicate that Cd027 diarrhea is uncommon in our area, and it appears mainly as imported cases. MLVA typing enables us to distinguish different genotypes among our Cd027 isolates.

Systematic use of universal 16S rRNA gene polymerase chain reaction (PCR) and sequencing for processing pleural effusions improves conventional culture techniques.

Conventional culture of pleural fluid samples frequently provides false-negative results. Universal polymerase chain reaction (PCR) of the 16S ribosomal ribonucleic acid (rRNA) gene (16S PCR) has proven useful in the diagnosis of various bacterial infections. We conducted a prospective study to assess the value of 16S PCR in the etiologic diagnosis of pleural effusion. All pleural fluid samples received for culture were also studied using 16S PCR. Positive samples were sequenced for identification. Clinical records and conventional culture results were analyzed to classify pleural fluid samples as infected or not infected. We studied 723 samples. We excluded 188 samples because they were obtained from a long-term chest tube, there was a diagnosis of mycobacterial infection, or there were insufficient data to classify the episode. Finally, 535 pleural fluid samples were analyzed. According to our criteria, 82 (15.3%) were infected and 453 (84.7%) were not infected. In the infected samples, 16S PCR was positive in 67 samples (81.7%) while conventional culture was positive in 45 (54.9%). There were 4 false positives with 16S PCR (0.9%) and 12 with culture (2.6%). The values for the etiologic diagnosis of bacterial pleural effusion of conventional culture compared with 16S PCR were as follows: sensitivity, 54.9%/81.7%; specificity, 97.4%/99.1%; positive predictive value, 76.3%/94.4%; negative predictive value, 92.6%/96.8%; and accuracy, 90.8%/96.5%.When compared with conventional culture, 16S PCR plus sequencing substantially improves the etiologic diagnosis of infectious pleural effusion. In our opinion, this technique should be added to the routine diagnostic armamentarium of clinical microbiology laboratories.