Diagnosis of bacteremia in whole-blood samples by use of a commercial universal 16S rRNA gene-based PCR and sequence analysis.

In a prospective, multicenter study of 342 blood samples from 187 patients with systemic inflammatory response syndrome, sepsis, or neutropenic fever, a new commercial PCR test (SepsiTest; Molzym) was evaluated for rapid diagnosis of bacteremia. The test comprises a universal PCR from the 16S rRNA gene, with subsequent identification of bacteria from positive samples by sequence analysis of amplicons. Compared to blood culture (BC), the diagnostic sensitivity and specificity of the PCR were 87.0 and 85.8%, respectively. Considering the 34 BC-positive patients, 28 were also PCR positive in at least one of the samples, resulting in a patient-related sensitivity of 82.4%. The concordance of PCR and BC for both positive and negative samples was (47 + 247)/342, i.e., 86.0%. In total, 31 patients were PCR/sequencing positive and BC negative, in whom the PCR result was judged as possible or probable to true bacteremia in 25. In conclusion, the PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Despite the indispensability of BC diagnostics, the rapid detection of bacteria by SepsiTest appears to be a valuable tool, allowing earlier pathogen-adapted antimicrobial therapy in critically ill patients.

Rapid diagnosis of candidaemia by real-time PCR detection of Candida DNA in blood samples.

This study prospectively evaluated an 18S rRNA gene-targeted real-time PCR approach in comparison with standard blood culture (BC) diagnostics for rapid diagnosis of candidaemia in a large study population of 384 patients, including 902 whole blood samples from 468 infectious episodes (IEs) of 329 adults and 55 children with haematological malignancies and various forms of immunodeficiency, and intensive care unit patients. Seven out of eight BC-proven cases (87.5 %) of candidaemia and seven out of twelve BC-positive samples (58.3 %) were positive by the Candida-specific PCR. A positive PCR result was also obtained for 28/460 BC-negative samples from IEs, including 8 patients with culture-confirmed Candida infection at primary sterile body sites. Of the PCR-positive, culture-negative patients, more than 50 % received systemic antifungal therapy. In 432/460 BC-negative IEs, the Candida specific-PCR was negative, resulting in a negative predictive value of 99.8 %. In conclusion, the Candida specific-PCR approach facilitates rapid detection of Candida DNA in blood samples of patients at risk of candidaemia within a few hours. Although standard BC diagnostics appear to remain indispensable for the detection of all cases of candidaemia, this PCR assay allowed the detection of candidaemia at a mean of 3 days earlier than BC diagnostics. Thus, it enables earlier antifungal therapy for patients with suspected candidaemia and may prevent further complications.

Rapid detection of pathogens in blood culture bottles by real-time PCR in conjunction with the pre-analytic tool MolYsis.

OBJECTIVES: Rapid detection of pathogens in blood from septic patients is essential for adequate antimicrobial therapy and prognosis of patients. Aim of this study is the acceleration of detection and identification of bacteria and fungi in blood cultures by molecular methods before positive signalling in an automated system. This would allow an earlier appropriate antimicrobial therapy and may improve the prognosis of septic patients. METHODS: Samples were analysed with an eubacterial real-time PCR assay that enables detection of bacterial DNA and simultaneous differentiation of Gram-positive and Gram-negative bacteria. In addition, genus- and species-specific real-time PCR assays were used. DNA preparation was performed with the new tool MolYsis. RESULTS: With the Gram-differentiating PCR assay bacteria were detectable in concentrations of 10-20 CFU per PCR reaction. A positive PCR result was achieved in samples taken from spiked blood culture bottles between 5.0 and 8.7h prior to positive signalling of the BACTEC system. We were able to identify the causative organism in 11 out of 18 culture-positive blood cultures from patients with septicaemia with an average of 10.7h prior to positive signalling. Out of 83 culture-negative bottles six samples showed a positive PCR result. CONCLUSION: PCR analysis in conjunction with MolYsis DNA preparation allows rapid detection of pathogens in blood culture samples. Thus, the approach may be a valuable supplemental tool for blood cultures in patients with suspicion of infection with slow-growing pathogens or serious clinical condition.

IgG reactivity against non-conformational NH-terminal epitopes of the desmoglein 3 ectodomain relates to clinical activity and phenotype of pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune disease caused by immunoglobulin G (IgG) autoantibodies against the desmosomal adhesion molecules, desmoglein (Dsg)3 and Dsg1. The aim of the study was to relate IgG reactivity of 123 PV sera and 40 control sera against NH(2)-terminal non-conformational epitopes of Dsg3 and Dsg1 with disease activity and clinical phenotype by enzyme-linked immunosorbent assay. The results show that (i) the overall reactivity and the titres of IgG reactive with the Dsg3 ectodomain, Dsg3(1-566), significantly correlated with the disease activity of the PV patients; (ii) IgG reactivity against the NH(2)-terminus of Dsg3, Dsg3(1-161), was associated with active PV while there was no direct correlation between the IgG titres and the disease activity; (iii) IgG reactivity against the NH(2)-terminus of Dsg3, Dsg3(1-161), was associated with mucosal and mucocutaneous PV; (iv) IgG titres against a small stretch of the NH(2)-terminus of Dsg3, Dsg3(25-88), were associated with active PV; and (v) IgG in the PV sera detected non-conformational epitopes in addition to the previously identified conformation-dependent epitopes of the Dsg3 and Dsg1 ectodomains.

[Excimer laser therapy of alopecia areata--side-by-side evaluation of a representative area]. / Die Excimer-Laser-Therapie der Alopecia areata--Halbseitige Evaluation eines repräsentativen Areals.

We report for the first time on hair regrowth in alopecia areata of the scalp achieved with the 308-nm xenon-chloride excimer laser in a prospective side-by-side trial. The alopecia areata had shown progression over a period of three years, and various treatments had not been effective. Out of a number of affected areas, one representative lesion was chosen; one half of it was treated, the other half remained untreated. After 27 sessions (200 - 4000 mJ/cm2, cumulative dose 52.6 J/cm2) over 3 months, only the treated area showed hair growth; which suggests that this was most probably not a spontaneous remission.