Evaluation of different real time PCRs for the detection of Pneumocystis jirovecii DNA in formalin-fixed paraffin-embedded bronchoalveolar lavage samples.

The presence of Pneumocystis jirovecii in fresh clinical materials can be detected by PCR with high sensitivity and is thus preferred over microscopic methods. However, fresh materials are not always available, and on formalin-fixed paraffin-embedded materials, PCR may result in reduced detection rates. In this study the diagnostic sensitivity of P. jirovecii real time PCR on DNA isolated from fresh bronchoalveolar lavage (BAL) samples versus that from matched FFPE derived DNA is analyzed. Our results indicate that when targeting a small DNA fragment P. jirovecii PCR can be performed on FFPE BAL samples with acceptable sensitivity (up to 83.3%). This is considerably higher than the 33.3% positives observed by classical staining of these samples.

Treatment with clarithromycin prior to coronary artery bypass graft surgery does not prevent subsequent cardiac events.

BACKGROUND: Recently, it has been suggested that Chlamydia pneumoniae possibly plays a possible role in the pathogenesis of atherosclerosis. We investigated whether treatment with clarithromycin prior to coronary artery bypass graft (CABG) surgery would prevent subsequent cardiovascular events and mortality. METHODS: Patients who were scheduled for CABG surgery were randomly assigned to receive either clarithromycin or placebo until the day of surgery in a double-blind trial. During the 2 years of follow-up, mortality and cardiovascular events were assessed. RESULTS: Follow-up at 2 years was achieved for 473 patients. The mean duration of treatment was 16 days. Patient characteristics at baseline were well balanced between the 2 treatment groups. Mortality was equal in the 2 groups: 10 (4.2%) of 238 patients in the clarithromycin group and 9 (3.8%) of 235 patients in the placebo group (relative risk, 1.10; 95% CI, 0.42-2.89; P=1.0). Also, there were no significant differences in the proportion of patients who experienced cardiovascular events during the follow-up period: 20 (8.4%) of 238 patients in the clarithromycin group and 19 (8.1%) of 235 patients in the placebo group (relative risk, 1.04; 95% CI, 0.55-1.98; P=1.0). The overall rate of such events was 58 (12.3%) of 473 patients. CONCLUSIONS: Treatment with clarithromycin in patients scheduled for CABG surgery did not reduce the subsequent occurrence of cardiovascular events or mortality during a 2-year follow-up period.

Evaluation of Borrelia real time PCR DNA targeting OspA, FlaB and 5S-23S IGS and Borrelia 16S rRNA RT-qPCR.

Borrelia burgdorferi non-sensu lato (s.l.) strains occurred in the Netherlands. A multiplex OspA, FlaB, IGS real time PCR was compared to 16S rRNA/rDNA RT-qPCR with lower average Cycle threshold (Ct) and LOD on strain dilutions. Multiplexing increased sensitivity on CSF samples (n=74), distinguishing B. burgdorferi s.l. from non-s.l. strains.

Indigenous Infection with Francisella tularensis holarctica in The Netherlands.

We report here the first case of indigenous tularemia detected in The Netherlands, a nonendemic country, since 1953. Whole genome DNA sequence analysis assigned the isolate BD11-00177 to the genomic group B.FTNF002-00, which previously has been exclusively reported from Spain, France, Italy, Switzerland, and Germany. The patient had not been abroad for years, which implies that this is an indigenous infection. The current case might predict an upcoming distribution of Francisella tularensis holarctica genomic group B.FTNF002-00 in Europe.

Draft genome sequence of Francisella tularensis subsp. holarctica BD11-00177.

Francisella tularensis is a facultative intracellular bacterium in the class Gammaproteobacteria. This strain is of interest because it is the etiologic agent of tularemia and a highly virulent category A biothreat agent. Here we describe the draft genome sequence and annotation of Francisella tularensis subsp. holarctica BD11-00177, isolated from the first case of indigenous tularemia detected in The Netherlands since 1953. Whole genome DNA sequence analysis assigned this isolate to the genomic group B.FTNF002-00, which previously has been exclusively reported from Spain, France, Italy, Switzerland and Germany. Automatic annotation of the 1,813,372 bp draft genome revealed 2,103 protein-coding and 46 RNA genes.

Effect of clarithromycin treatment on Chlamydia pneumoniae in vascular tissue of patients with coronary artery disease: a randomized, double-blind, placebo-controlled trial.

Several small clinical trials have indicated that antibiotic treatment of Chlamydia pneumoniae infection is associated with a better outcome in patients with coronary artery disease (CAD). It has not been demonstrated whether antibiotic treatment eradicates C. pneumoniae from vascular tissue. The aim of the present study was to assess the effect of clarithromycin on the presence of C. pneumoniae in the vascular tissue of patients with CAD. Patients who had CAD and who were waiting for coronary artery bypass graft surgery were enrolled in a randomized, double-blind, placebo-controlled trial. Patients were treated with clarithromycin at 500 mg or placebo once daily from the day of inclusion in the study until surgery. Several vascular tissue specimens were obtained during surgery. The presence of C. pneumoniae in vascular tissue specimens was examined by immunohistochemical staining (IHC) and two PCR assays. Chlamydia immunoglobulin G (IgG) titers were determined by an enzyme-linked immunosorbent assay at the time of inclusion in the study and 8 weeks after surgery. A total of 76 patients were included, and 180 vascular tissue specimens were obtained (80 specimens from the group treated with clarithromycin and 100 specimens from the group treated with placebo). Thirty-five patients received clarithromycin (mean duration, 27 days; standard deviation [SD], 12.2 days), and 41 patients received placebo (mean duration, 27 days; SD, 13.9 days). IHC detected the C. pneumoniae major outer membrane protein antigen in 73.8% of the specimens from the group treated with clarithromycin and 77.0% of the specimens from the group treated with placebo (P was not significant). Chlamydia lipopolysaccharide antigen was found in only one specimen from the group that received placebo. C. pneumoniae DNA was not detected in any specimen. Baseline Chlamydia-specific IgG titers were equally distributed in both groups and were not significantly different after treatment. There was no indication of an active C. pneumoniae infection in vascular tissue. Chlamydia-specific IgG titers remained unchanged throughout the study in both the antibiotic- and the placebo-treated patients.

Impact of serological methodology on assessment of the link between Chlamydia pneumoniae and vascular diseases.

We assessed the impact of five serologic tests on the link between Chlamydia pneumoniae and abdominal aortic aneurysms (AAA). The results of the tests were inconsistent. Agreement among the five tests was generally poor. Detection of the link between C. pneumoniae and AAA depends on the serologic methodology chosen.

Is the perceived association between Chlamydia pneumoniae and vascular diseases biased by methodology?

Inter- and intralaboratory inconsistencies in detection rates of Chlamydia pneumoniae in vascular specimens have been demonstrated. In this study, 66 vascular tissue specimens from 66 patients with vascular disease were tested by three PCR assays: a 16S PCR-based reverse line blot (RLB) assay, a single-step PCR, and a nested PCR. Also, we explored the impacts of different DNA polymerase enzymes on the results based on gel electrophoresis and hybridization. The PCR results by gel electrophoresis in the single-step PCR depended on which DNA polymerase was used. All samples were negative with AmpliTaq Gold DNA polymerase, and 54.5% (36 of 66) were positive with the conventional Taq DNA polymerase. All samples were negative after hybridization with a C. pneumoniae-specific probe. In the nested PCR, all specimens were negative by gel electrophoresis and after hybridization. The RLB assay failed to detect C. pneumoniae in any specimen; however, 20 specimens were Chlamydia sp. positive. The sequence analysis of six of these samples demonstrated Chlamydia-like organisms. RLB detected Chlamydia sp. DNA in water and in the elution buffer after passage of the Qiagen columns (11 of 40). This study identified factors that may influence the detection of C. pneumoniae DNA in vascular tissues and consequently bias the perception of a link between C. pneumoniae and vascular diseases. The following are strongly recommended: to use DNA polymerases that have to be activated, to decontaminate with dUTP-uracil-DNA glycosylase, to hybridize with specific probes, to include sufficient controls, and to use molecular grade water.

Effect of clarithromycin on inflammatory markers in patients with atherosclerosis.

Atherosclerosis can to a certain extent be regarded as an inflammatory disease. Also, inflammatory markers may provide information about cardiovascular risk. Whether macrolide antibiotics, especially clarithromycin, have an anti-inflammatory effect in patients with atherosclerosis is not exactly known. To study this phenomenon, a placebo-controlled, randomized, double-blind study was performed. A total of 231 patients with documented coronary artery disease received a daily dose of either 500 mg of slow-release clarithromycin or placebo until the day of surgery. Levels of inflammatory markers (C-reactive protein, interleukin-2 receptor [IL-2R], IL-6, IL-8, and tumor necrosis factor alpha) were assessed during the preoperative outpatient visit, on the day of surgery, and 8 weeks after surgery. Also, changes in the levels of inflammatory markers between visits were determined by delta calculations. Baseline patient characteristics were balanced between the two treatment groups: the average age was 66 years (standard deviation [SD] = 9.0), 79% of the patients were male, and the average number of tablets used was 16 (SD = 9.3). The inflammatory markers of the groups as well as the delta calculations were not significantly changed. Treatment with clarithromycin did not influence the inflammatory markers in patients with atherosclerosis.

Mycobacterium heckeshornense infection in an immunocompetent patient and identification by 16S rRNA sequence analysis of culture material and a histopathology tissue specimen.

Mycobacterium heckeshornense is a rare isolate in clinical specimens. We performed simultaneous 16S rRNA sequence analysis of a mycobacterium culture and a histopathology specimen to determine the relevance of M. heckeshornense infection in an immunocompetent patient initially presenting with pneumothorax.