Annika Pettersson

Development of an internally controlled PCR assay for broad range detection of bacteria in platelet concentrates.

A real-time PCR assay based on the 16S rRNA gene was optimized for the detection of a broad range of bacteria in plasma and platelet concentrates (PC). A lambda phage internal control was constructed and implemented in the assay, which made it suitable for diagnostic use. Spiking studies in plasma and PCs were performed to determine the analytical sensitivity of the assay. Thirty three colony forming units (CFU)/ml of E. coli and 72 CFU/ml of Staphylococcus epidermidis could be detected in plasma, and 97 CFU/ml of S. epidermidis in PCs. The assay detected all bacteria relevant for bacterial contamination of PCs. The short turn around time of the assay made it suitable for testing PCs for bacterial contamination prior to transfusion.

Trends in Extended Spectrum Beta-Lactamase (ESBL) Producing Enterobacteriaceae and ESBL Genes in a Dutch Teaching Hospital, Measured in 5 Yearly Point Prevalence Surveys (2010-2014)

This paper describes the trends in prevalence of ESBL producing Enterobacteriaceae (ESBL-E) and ESBL genes, measured in five consecutive yearly Point Prevalence Surveys (PPS). All patients present in the hospital and in a day-care clinic (including patients on dialysis) on the day of the survey, were screened for perianal ESBL-E carriage. Perianal swabs were taken and cultured using an enrichment broth and a selective agar plate. Both phenotypic and genotypic methods were used to detect the production of ESBL, presence of ESBL-genes and clonal relatedness. Out of 2,695 patients, 135 (5.0%) were tested ESBL-E positive. The overall ESBL-E prevalence was stable over the years. Overall 5.2% of all ESBL-E were acquired by nosocomial transmission. A relative decrease of CTX-M-1-1-like ESBL genes (from 44 to 25%, p = 0.026) was observed, possibly related to the strong (>60%) decrease in antibiotic use in livestock in our country during the same period.

Distribution, origin and contamination risk of coagulase-negative staphylococci from platelet concentrates.

Transfusion-associated bacterial sepsis is the most common microbiological risk of transfusion and is caused mostly by platelet concentrates (PCs). The most frequently identified bacterial contaminants of PCs are coagulase-negative staphylococci (CNS). In order to learn more about the distribution, source and risk of the CNS that are involved in bacterial contamination of PCs, CNS strains isolated during platelet screening were collected and characterized to the species level with three different methods: 16S rRNA and sodA gene sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and amplified fragment length polymorphism (AFLP) analysis. AFLP analysis was also used for the typing of the CNS strains. A total of 83 CNS strains were analysed by sequencing and 8 different CNS species were identified, with Staphylococcus epidermidis being the predominant species. MALDI-TOF MS and AFLP analysis confirmed these results to a large extent. However, MALDI_TOF MS could not identify all strains to the species level and AFLP analysis revealed an additional, likely novel, CNS species. The species identified are mainly recognized as being part of the normal skin flora. Typing of the CNS strains by AFLP analysis showed that there was not a unique strain which is significantly more often present during bacterial contamination of PCs.

Development of a reverse transcription–polymerase chain reaction assay for eubacterial RNA detection in platelet concentrates

BACKGROUND: The sensitivity of a real‐time polymerase chain reaction (PCR) assay detecting bacteria in platelet concentrates (PCs) was improved by detection of ribosomal RNA (rRNA) in addition to DNA. The real‐time reverse transcription–PCR (RT‐PCR) assay was compared with the BacT/ALERT culturing system (bioMérieux) to determine its value for routine screening of PCs for bacterial contamination.

Performance and suitability of polymerase chain reaction for early detection of bacteria in platelet concentrates.

BACKGROUND: In this study the applicability of a 16S rRNA real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) and a Staphylococcus genus–specific PCR for screening of bacterial contamination in platelet concentrates (PCs) was determined.

Clinical correlates of herpes simplex virus type 1 loads in the lower respiratory tract of critically ill patients

BACKGROUND The significance of isolation of herpes simplex virus (HSV) type 1 from the lower respiratory tract in critically ill patients on mechanical ventilation is still unclear. In the current study, we used polymerase chain reaction techniques to quantify HSV-1 to further evaluate its role. OBJECTIVES The hypothesis was that high loads reflect invasive pulmonary disease related to prolonged mechanical ventilation and increased mortality, as opposed to shedding from the upper respiratory tract, which leads to lower viral loads. STUDY DESIGN We prospectively studied 77 consecutive patients admitted to the intensive care unit and analyzed 136 tracheal aspirates or bronchoalveolar lavage fluids, taken when clinically indicated in the diagnostic workup of fever, radiologic pulmonary infiltrates, progressive respiratory insufficiency or combinations. Samples were cultured for bacteria and yeasts according to routine microbiological methods and HSV-1 loads were determined by real time quantitative PCR. Viral loads were expressed per number of cells recovered. RESULTS HSV-1 load was directly related to the simplified acute physiology score II (rs=0.47, P=0.04) when the first specimen taken proved positive for HSV-1. HSV-1 positivity concurred with Candida spp. colonization. Patients with and without a HSV-1 load did not differ with respect to pulmonary and systemic courses and vital outcomes. CONCLUSIONS The data suggest that HSV-1 in the lower respiratory tract originates from shedding in the upper respiratory tract in about 30% of critically ill patients, following immune suppression and reactivation, without invasively infecting the lung. No attributable mortality was observed.

Gastroenteritis caused by Campylobacter concisus.

We describe a case of gastroenteritis caused by Campylobacter concisus. The pathogenic potential of C. concisus has yet to be elucidated. Recent studies indicate an association with enteric disease in immunocompromised patients and inflammatory bowel disease in children. Molecular identification methods may be necessary for identifying certain Campylobacter species because of phenotypic similarity.

Molecular relatedness of Propionibacterium species isolated from blood products and on the skin of blood donors

BACKGROUND: In this study it was investigated whether Propionibacterium acnes present in platelet concentrates (PCs) and related red blood cells (RBCs), originate from the skin of the donor.

Reducing the Risk of Transfusion-Transmitted Bacterial Infections in Platelet Concentrates: Current Status and Developments

Bacterial contamination of blood products is currently one of the greatest microbiological risks of transfusion. Of all blood products, platelet concentrates (PCs) are most frequently implicated in transfusiontransmitted bacterial infections. Several methods have been developed to reduce this risk. The most frequently used method at the moment is a culturing system, the BacT/ Alert (bioMerieux, Boxtel, The Netherlands). Although the method is sensitive, it also has several drawbacks. Therefore, new methods are being developed to increase the safety of transfusion of platelet concentrates in the future.

Tropheryma whipplei, a Potential Commensal Detected in Individuals Undergoing Routine Colonoscopy.

ABSTRACT Mucosal biopsy samples from individuals not suspected of having Whipples disease were tested for the presence of Tropheryma whipplei. A sensitive and specific real-time PCR assay targeting a sequence present seven times in the T. whipplei genome was used. T. whipplei DNA was detected in 2.0 and 3.8% of the patients undergoing gastroduodenoscopy and colonoscopy, respectively, who were tested.