Måns Ullberg

Clinical Evaluation of the FilmArray Blood Culture Identification Panel in Identification of Bacteria and Yeasts from Positive Blood Culture Bottles

ABSTRACT The FilmArray platform (FA; BioFire, Salt Lake City, UT) is a closed diagnostic system allowing high-order multiplex PCR analysis with automated readout of results directly from positive blood cultures in 1 h. In the present study, we evaluated the clinical performance of the FilmArray blood culture identification (BCID) panel, which includes 19 bacteria, five yeasts, and three antibiotic resistance genes. In total, 206 blood culture bottles were included in the study. The FilmArray could identify microorganisms in 153/167 (91.6%) samples with monomicrobial growth. Thirteen of the 167 (7.8%) microorganisms were not covered by the FilmArray BCID panel. In 6/167 (3.6%) samples, the FilmArray detected an additional microorganism compared to blood culture. When polymicrobial growth was analyzed, the FilmArray could detect all target microorganisms in 17/24 (71%) samples. Twelve blood culture bottles that yielded a positive signal but showed no growth were also negative by FilmArray. In 3/206 (1.5%) bottles, the FilmArray results were invalid. The results of the FilmArray were reproducible, as demonstrated by the testing and retesting of five bottles in the same day and a longitudinal follow-up of five other blood cultures up to 4 weeks. The present study shows that the FilmArray is a rapid identification method with high performance in direct identification of bacteria and yeasts from positive blood culture bottles.

Purification and characterisation of a plasminogen-binding protein from Haemophilus influenzae. Sequence determination reveals identity with aspartase.

Plasminogen binding proteins have been described both for Gram positive and Gram negative bacteria. In the present work we describe the purification and characterization of a plasminogen binding protein from Haemophilus influenzae (strain HI-23459). Bacteria were sonicated in order to solubilize plasminogen-binding proteins. The supernatant was subjected to affinity chromatography on plasminogen kringle-4 fragment bound to Sepharose 4B and subsequently processed by ion-exchange chromatography on DEAE-Sepharose CL-6B. Characterization of the protein by SDS-PAGE displayed a single band with a molecular mass of about 55,000, both prior to and after reduction. The purified protein stimulates tPA (tissue plasminogen activator) catalysed plasminogen activation by a factor of approximately 300, mainly due to a decrease in K(m). Antibodies were raised in rabbits and used in quantitative and qualitative analysis. However, using a FITC-conjugate we failed to demonstrate the presence of the purified protein on the surface of intact bacteria. The corresponding gene was isolated from a lambda EMBL3 phage library prepared from chromosomal DNA from the same H. influenzae strain, using an oligonucleotide probe based on the NH2-terminal amino acid sequence. An open reading frame corresponding to 472 amino acid was found. The amino acid sequence of the translated gene demonstrates 97% identity with the recently published sequence from aspartate ammonia lyase (aspartase) from H. influenzae. Enzymatic analysis of the purified protein revealed a high aspartase activity.

Identification of 43 Streptococcus Species by Pyrosequencing Analysis of the rnpB Gene

ABSTRACT Pyrosequencing technology was evaluated for identification of species within the Streptococcus genus. Two variable regions in the rnpB gene, which encodes the RNA subunit of endonuclease P, were sequenced in two reactions. Of 43 species, all could be identified to the species level except strains of the species pairs Streptococcus anginosus/S. constellatus and S. infantis/S. peroris. A total of 113 blood culture isolates were identified by pyrosequencing analysis of partial rnpB sequences. All but eight isolates could be unambiguously assigned to a specific species when the first 30 nucleotides of the two regions were compared to an rnpB database comprising 107 streptococcal strains. Principal coordinate analysis of sequence variation of strains from viridans group streptococci resulted in species-specific clusters for the mitis and the salivarius groups but not for the anginosus group. The identification capacity of pyrosequencing was compared to the biochemical test systems VITEK 2 and Rapid ID 32 Strep. The concordance between pyrosequencing and VITEK 2 was 75%, and for Rapid ID 32 Strep the corresponding figure was 77%. Isolates with discrepant identifications in the three methods were subjected to entire rnpB DNA sequence analysis that confirmed the identifications by pyrosequencing. In conclusion, pyrosequencing analysis of the rnpB gene can reliably identify Streptococcus species with high resolution.

New receptor for human plasminogen on gram positive cocci

180 bacterial strains representing 17 different species of gram positive cocci were tested for the ability to interact with human plasminogen. Receptors for plasminogen could be detected on 23/24 strains of S. pyogenes, 15/15 strains of S. equisimilis, 14/16 strains of human group G streptococci and 14/14 strains of S. pneumoniae. Eight of nineteen strains representing five species of alpha‐hemolytic streptococci were also positive. S. equisimilis demonstrated the highest uptake with a median value of 58 per cent (20%‐67%). On the other hand, all strains of S. agalactiae, the majority of S. faecalis and all S. aureus, S. epidermidis and S. saprophyticus strains tested were negative. The concentration of unlabelled plasminogen causing a 50 per cent reduction of bound tracer was between 50 and 150 nM. These estimates of the dissociation constant confirmed the specific nature of the interaction. Binding of plasminogen could be blocked by addition of plasmin‐aprotinin complex, suggesting that plasminogen and plasmin bind to the same receptor. Binding was also blocked by the plasminogen fragment kringle 1–3, but not by miniplasminogen, a fragment containing kringle 5 and the B‐chain region. As streptokinase interacts mainly with the B‐chain of plasmin it is clear that the bacterial receptor for plasminogen is not identical to streptokinase.

Multiplex Real-Time PCR Targeting the RNase P RNA Gene for Detection and Identification of Candida Species in Blood

ABSTRACT We have developed a single-tube multiplex real-time PCR method for the detection of the eight most common Candida species causing septicemia: Candida albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, and C. tropicalis. The method developed targets the RNase P RNA gene RPR1. Sequences of this gene were determined for seven of the Candida species and showed surprisingly large sequence variation. C. glabrata was found to have a gene that was five times longer gene than those of the other species, and the nucleotide sequence similarity between C. krusei and C. albicans was as low as 55%. The multiplex PCR contained three probes that enabled the specific detection of C. albicans, C. glabrata, and C. krusei and a fourth probe that allowed the general detection of the remaining species. The method was able to detect 1 to 10 genome copies when the detection limit was tested repeatedly for the four species C. albicans, C. glabrata, C. krusei, and C. guilliermondii. No significant difference in the detection limit was seen when the multiplex format was compared with single-species PCR, i.e., two primers and one probe. The method detected eight clinically relevant Candida species and did not react with other tested non-Candida species or human DNA. The assay was applied to 20 blood samples from nine patients and showed a sensitivity similar to that of culture.

Interferon-induced NK augmentation in humans. An analysis of target recognition, effector cell recruitment and effector cell recycling.

By combining a single‐cell cytotoxicity assay in agarose with estimations of the maximal natural killer (NK) cell potential (Vmax) by 51Cr release, the mechanisms behind interferon augmentation of human NK cells were analysed. The number of target‐binding cells (TBCs) the fraction of active TBCs, and NK cell recycling were studied after short‐term interferon treatment. The results demonstrate a dual effect of interferon on human NK cells: effector cell recruitment and increased effector cell recycling. Both of these variables were increased when NK cells were tested against the standard target K‐562 and against Daudi and BJAB cells, derived from B‐type lymphomas. However, when T cell lines derived from acute lymphocytic leukaemia (Molt‐4 and 1301) were used as targets, a larger fraction of active NK cells were found among untreated TBCs, whereas inteferon treatment only resulted in increased effector cell recycling and not in effector cell recruitment. No increase in TBCs after interferon treatment could be detected with any cell line tested. The difference seen between T and non‐T cell lines with regard 10 interferon‐induced effector cell recruitment is discussed in relation to known characteristics of the human NK system.

Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout

A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.

Two types of receptors for human plasminogen on group G streptococci

To investigate the nature of plasminogen binding to streptococci, strains selected for high reactivity with human plasminogen were examined for binding pattern against a panel of plasminogen fragments. The strains included human isolates of groups A, C and G as well as bovine isolates of group G. All strains reacted substantially with the plasminogen fragment kringle 1–3. Using the miniplasminogen fragment (kringle 5 and the B chain) a small but reproducible uptake was detected for human group G strains but not for group A or C strains. The group G strains of bovine origin on the other hand demonstrated high uptake of miniplasminogen, suggesting the possibility of an alternative plasminogen receptor for this species. This interpretation was supported by blocking experiments with the lysine analogue EACA where low concentrations (1 mM) completely blocked plasminogen binding to human streptococci, whereas a 100‐fold higher concentration was needed for bovine group G strains. Scatchard plots with human isolates resulted in straight lines and Kd values were generally in the range of 20–80 nM. The number of receptors was estimated to be 45,000 for a selected group A strain and about 10,000 for the selected group C and G strains. Scatchard analysis with bovine group G isolates on the other hand revealed a two phase interaction, supporting the assumption of two different receptor structures on these strains. Kd for the first phase was estimated to be about 20 nM (10,000–20,000 receptors per bacterium), which was similar to the human strains, whereas the second phase was in the range of 400–500 nM (50,000 and 150,000 receptors per bacterium with two selected strains). Scatchard plots with the miniplasminogen fragment as ligand mimicked the phase two reaction with plasminogen, supporting the concept that this reaction represents a new and not previously described receptor. Both the receptor reacting with the kringle 1–3 portion and the one reacting with the miniplasminogen portion bound plasmin and plasminogen with similar affinity.

Inhibition of Human NK Cell Cytotoxicity by Induction of Cyclic AMP Depends on Impaired Target Cell Recognition

Induction of cyclic AMP (cAMP) depresses natural killer (NK) cell activity. The present results demonstrate that this is dependent on a decreased capacity of the effector cells to conjugate to target cells. This was found either if dibutyryl‐cAMP was used or if cAMP was induced by adenylate cyclase stimulation with prostaglandin E1 (PGE1) or by inhibition of phosphodiesterase activity with the inhibitor ZK 62711. The sites of action for cAMP‐induced NK suppression and Interferon (IFN)‐induced NIC enhancement are demonstrated to be distinct, since IFN acts by increasing the lytic efficiency and the recycling capacity without influencing target binding. Sequential treatment with cAMP/JFN and JFN/cAMP shows that IFN can neither restore target binding when added after cAMP nor protect against the cAMP‐induced target binding inhibition when added before cAMP. The results are discussed in view of earlier data on cAMP in relation to cell membrane functions and cellular recognition, the mechanism underlying the cAMP‐induced target binding inhibition, and the potential of the NK system as an indicator for immunosuppression. The present work also demonstrates the particular subpopulation in peripheral blood which mediates most NK activity, to respond strongly to PGEi stimulation with regard to cAMP induction.

Clinical comparison of the Bactec Mycosis IC/F, BacT/Alert FA, and BacT/Alert FN blood culture vials for the detection of candidemia

The present study analyzed the performance of Bactec Mycosis IC/F, BacT/Alert FA, and BacT/Alert FN vials in detection and time to detection (TTD) of Candida spp. in 179 simultaneous blood cultures. The Mycosis IC/F, BacT/Alert FA, and BacT/Alert FN vials could detect Candida spp. in 144 (80.45%) of 179, 149 (83.24%) of 179, and 8 (4.47%) of 179 samples, respectively. With the presence of antifungal therapy, the numbers of positive vials were higher in BacT/Alert FA compared to Mycosis IC/F, 87/99 versus 73/99, respectively (P < 0.05). TTD (SD) for C. albicans was shorter in Mycosis IC/F than in BacT/Alert FA vials without antifungal therapy, 20.89 (9.33) versus 28.26 (9.77), respectively (P < 0.01). The detection of Candida spp., with concomitant bacteremia, was higher in Mycosis IC/F than in BacT/Alert FA vials, 28/30 and 19/30, respectively (P = 0.01). The present data show that the use of Bactec Mycosis IC/F together with BacT/Alert FA vials might improve the detection of Candida spp.