Evaluation of six PCR assays in combination with patient related data for the diagnosis of Clostridium difficile-associated infections.

BACKGROUND: The aim of this multicenter study was to establish a diagnostic algorithm using molecular methods for the diagnosis of C. difficile-associated infection (CDI). In addition patient specific data were taken into consideration for the interpretation of the results. METHODS: We compared the performance of six different commercially available PCR-tests, two toxin immunoassays, and a glutamat-dehydrogenase test by analysing liquid stool specimens from patients with suspected CDI. Toxigenic culture on CLO-agar was used as reference method. RESULTS: In total 250 stool specimens were collected at two study sites. 77 (30.8%) stool samples were culture-positive for toxigenic C. difficile. 173 (69.2%) specimens showed no growth of C. difficile. As a result, each of the PCR assays tested for C. difficile had a significantly higher sensitivity (94.8% - 100%) and NPV (97.6% - 100%) than the TOX-EIA with a sensitivity of 57.1% and NPV of 83.8%. Specificity of the PCR tests was 94.1% to 96.0% and PPV between 86.5% and 91.6%. The analysis of the patient data revealed a significant difference (p-value 0.0202) between toxin-positive and toxin-negative patients regarding prior antibiotic treatment, especially for cephalosporins. CONCLUSIONS: Our findings support the recommendation to restrict the use of antibiotics as a cornerstone in the prevention of CDI. We conclude that all of the PCR assays evaluated in this study can be applied in a diagnostic algorithm.

Evaluation of the FluoroType MTB assay for the rapid and reliable detection of Mycobacterium tuberculosis in respiratory tract specimens.

We performed the first evaluation study of the new FluoroType MTB assay, a HyBeacon based nucleic acid amplification test for the direct detection of Mycobacterium tuberculosis in respiratory tract specimens. Using mycobacterial culture as a reference, 661 specimens were tested. Sensitivity and specificity were 95.1% and 96.4% for all specimens, sensitivity was 100% for smear-positive and 84.6% for smear negative specimens, respectively. The FluoroType MTB assay showed fast and accurate results within 3 hours, including DNA extraction.

[Severe pneumococcal infections in the elderly - preventable by vaccination]. / Schwere Pneumokokkeninfektionen im Alter - impfpräventabel.

Elderly people are more susceptible to pneumococcal infections. Data in Germany from 2005-2010 shows that especially seniors are prone to develop serious complications such as sepsis. Women are obviously less affected than men. Most of the infections occurred during the winter months. The majority of isolates, i.e., about 80%, possess capsular polysaccharide antigens which are represented in the 23-valent vaccine. Consequently, it could be assumed that the severe complications ensuing long hospital stays and associated with a high mortality could have been avoided, if the elderly people would have been vaccinated, which, however, was only true in a small proportion (28%). Recently, a new conjugated vaccine was introduced to the market. In principle, several antibiotics are available for direct antibacterial treatment. All isolates are susceptible to cefotaxime as well as to ceftriaxone. Resistance to penicillin as well as ampicillin is very rare in Germany. The vast majority of isolates are susceptible to quinolones such as levofloxacin and moxifloxacin. Resistance to macrolides, for example to erythromycin, occurs to a certain extent but the percentage has been declining in recent years. Nevertheless, in many instances therapy is too late. Thus, prevention is of great importance.

Evaluation of a new real-time PCR assay for the direct detection of diarrheagenic Escherichia coli in stool specimens.

Diarrheagenic E. coli (DEC) are one of the most common causes for diarrhea worldwide, especially in children. We evaluated the rapid RIDA ® GENE (RG) real-time multiplex PCR assays (R-Biopharm, Darmstadt, Germany) for the detection of the most important diarrheagenic E. coli. Three hundred fifteen liquid or non-formed stool specimens were examined. The results of the RG multiplex assays were compared to specific PCR methods. The sensitivity and specificity of the RG PCRs were as follows, 100%/100% for the detection of EHEC, 96.3% and 99% for EPEC, 100% and 100% for the detection of EAEC, ETEC and EIEC, respectively. Overall, the RG real-time PCR system for the detection of DEC tested in this study provided reliable and rapid results and shows the ability as a useful addendum for the detection of diarrheagenic E. coli in the medical laboratory.

Direct detection of methicillin-resistant Staphylococcus aureus in clinical specimens by a nucleic acid-based hybridisation assay.

The occurrence of methicillin-resistant Staphylococcus aureus (MRSA) is still increasing worldwide and is associated with significant morbidity, mortality and hospital costs. Screening for MRSA plays a key role in limiting further nosocomial spread of this organism. Control measures require a rapid and sensitive test for direct detection of MRSA carriage. This study evaluated an easy-to-use PCR-hybridisation assay for the direct detection of MRSA in clinical swab specimens. In total, 508 pairs of swabs from 242 patients at risk for MRSA carriage were analysed by the standard culture method and the PCR assay. One swab was used for PCR and culture, while the second was used for culture only. Of the 508 pairs tested, 37 were positive by culture and 35 were positive by PCR. Among the 471 culture-negative specimens, 465 were negative by PCR and six were PCR-positive. The PCR assay had a sensitivity of 94.59%, a specificity of 98.73%, a positive predictive value of 85.37%, and a negative predictive value of 99.57%. The PCR-hybridisation assay enabled reliable detection of MRSA carriage in c. 4 h, thereby allowing its effective use in an MRSA control strategy.

[Laboratory survey on the incidence of Pneumocystis jirovecii - obviously a peculiar fungus, but also a rare pathogen?]. / Laborerhebung zur Häufigkeit von Pneumocystis jirovecii - Zwar ein besonderer Pilz, aber auch ein seltener Erreger?

Pneumocystsis jirovecii is a peculiar fungus for a variety of reasons. This opportunistic pathogen multiplies in humans only under certain conditions; a defect in the T-cell defense system creates a predisposition to this infection. In 2010 a data survey (IFT as well as PCR) from a few laboratories in Germany revealed 412 positive individuals. Even if only a few patients test positive for the colonization stage of this pathogen, the sheer number of individuals testing positive for other stages of infection indicate that the incidence of pneumocystosis in immunocompromised patients in Germany is underestimated.

[Pneumococcal sepsis in aged patients - implications for the vaccination of seniors against pneumococci]. / Pneumokokkensepsis im Alter - Implikationen für die Impfung alter Menschen gegen Pneumokokken.

In the years 2005-2010 pneumococci were isolated in the Limbach laboratory/Heidelberg from blood cultures of 1,085 patients. Obviously, older patients are more prone to these bacteria, since 66 % of the patients were older than 60 years. All isolates were susceptible to cefotaxime; 3 % of isolates were resistant to penicillin, 2 % were resistant to levofloxacin and 15 % were resistant to erythromycin. From 457 out of the isolates serotyping was achieved: more than 80 % of the isolates were covered by the 23-valent vaccine. This means that particularly old people should be vaccinated against pneumococci, because they will profit probably most from such preventive measurements.

[Interventional ERCP in patients with cholestasis. Degree of biliary bacterial colonization and antibiotic resistance]. / Interventionelle ERCP bei Patienten mit Cholestase: Häufigkeit und Antibiotikaresistenz der biliären Keimbesiedlung.

Interventional ERCP in patients with cholestasis. Degree of biliary bacterial colonization and antibiotic resistance. Biliary obstruction together with bacterial colonization of the bile duct may lead to development of acute cholangitis. The aim of our prospective study was to investigate the presence and degree of biliary bacterial colonization by means of bile aspiration during ERCP in patients with biliary obstruction. Furthermore, we evaluated antibiotic therapy regimens, which would cover the bacterial species obtained by ERCP and subsequent culture in each patient. In addition, analysis of risk factors was performed that would predispose to the development of cholangitis.80 patients with clinical and laboratory evidence of biliary obstruction underwent ERCP with initial aspiration of bile via the cannulation catheter. This material was used to culture aerobic and anaerobic bacteria and determine the colony count/ml bile, followed by identification of each species and antibiotic resistance testing. The minimal inhibitory concentration for Levofloxacin, Ciprofloxacin, Piperacillin, Ampicillin, Ceftriaxone, Imipenem, Gentamycin und Metronidazole was determined. Immediately after the ERCP or if the body temperature (after ERCP) rose to > 38 degrees C blood cultures were obtained. In 45 (56 %) patients biliary colonization with bacteria could be identified (56 %). In 20 patients a single isolate was cultured, in 25 cases mixed infection was present. A total of 83 species were isolated. The most common bacteria were E. coli, Enterococcus and Klebsiella. 9.6 % of all isolates were obligatory anaerobes. In 9 of 80 patients bloodcultures tested positive for bacterial growth (rate of bacteremia: 11.3 %). 10 patients had acute cholangitis clinically before ERCP, 13 patients developed signs of infection after ERCP. Statistically significant factors contributing to the risk of infection were age of the patient, the clinical condition of the patient before ERCP and the biliary colony count. Patients with development of infection after ERCP showed a significantly higher incidence of bacterial colonization of the biliary tree and a higher colony count. In all bacterial species Imipenem (4.5 %) or Levofloxacin (2.2 %) exhibited the lowest rate of in-vitro resistance. Based on these data, the implementation of Levofloxacin in combination with anaerobic coverage is advantageous as a calculated therapy for patients with acute cholangitis.