Christiane Beckmann

Diagnostic performance of near-patient testing for influenza

Abstract Background Rapid diagnosis of influenza is important for controlling outbreaks and starting antiviral therapy. Direct antigen detection (DAD) is rapid, but lacks sensitivity, whereas nucleic acid amplification testing (NAT) is more sensitive, but also more time-consuming. Objectives To evaluate the performance of a rapid isothermal NAT and two DADs. Study design During February–May 2014, we tested 211 consecutive patients with influenza-like illness using a commercial isothermal NAT (Alere™ Influenza A&B) as well as the DAD Sofia® Influenza A+B and BinaxNOW® Influenza A&B for detection of influenza-A and -B virus. RespiFinder-22® a commercial multiplex NAT served as reference test. Serial 10-fold dilutions of influenza-A and -B cell culture supernatants were examined. Another 225 patient samples were tested during December 2014–February 2015. Results Compared to RespiFinder-22®, the isothermal NAT Alere™ Influenza A&B, and the DAD Sofia® Influenza A+B and BinaxNOW® Influenza A&B had sensitivities of 77.8%, 59.3% and 29.6%, and specificities of 99.5%, 98.9% and 100%, respectively, for the first 211 patient samples. Alere™ Influenza A&B showed 85.7% sensitivity and 100% specificity in the second cohort. Isothermal NAT was 10-100-fold more sensitive compared to DAD for influenza virus culture supernatants with a lower limit of detection of 5000–50,000 copies/mL. The average turn-around time (TAT) of isothermal NAT and DADs was 15min, but increased to 110min for Alere™ Influenza A&B, 30min for BinaxNOW® Influenza A&B, and 45min for Sofia® Influenza A+B, when analyzing batches of 6 samples. Conclusion Simple sample processing and a TAT of 15min render isothermal NAT Alere™ Influenza A&B suitable for sequential near-patient testing, but the TAT advantage is lost when testing of larger series.

Comparing Luminex NxTAG-Respiratory Pathogen Panel and RespiFinder-22 for multiplex detection of respiratory pathogens.

Respiratory tract infection (RTI) involves a variety of viruses and bacteria, which can be conveniently detected by multiplex nucleic acid amplification testing (NAT). To compare the novel Luminex‐based NxTAG‐Respiratory Pathogen Panel (NxTAG‐RPP) with the routine multiplex‐ligation‐NAT based RespiFinder‐22® (RF‐22), 282 respiratory specimens including nasopharyngeal swabs (71%), broncho‐alveolar lavage (27%), throat swabs, tracheal secretions, and sputum (2%) from 116 children and 155 adults were extracted using a Corbett CAS1200 (Qiagen), and analyzed in parallel by the routine RF‐22 and NxTAG‐RPP. Concordant results were obtained in 263 (93.3%) cases consisting of concordant positives in 167 (59.2%) and concordant negatives in 96 (34%). Results were discordant in 19 (6.7%) consisting of 15 positive:negative, and 4 negative:positive results by NxTAG‐RPP versus RF‐22, respectively. Co‐infections were observed in 10.3% with NxTAG‐RPP and in 5.9% with RF‐22. Most additional viral pathogens identified by the NxTAG‐RPP involved dual infections with rhinovirus and RSV. Discordant samples were mainly due to low genome signals of Ct less than 36, when retested by QNAT suggesting a higher sensitivity of the NxTAG‐RPP, also when detecting multiple infections. Hands‐on time after extraction for 24 and 96 samples was 0.25 and <0.5 hr for the NxTAG‐RPP, and 2 and 4 hr for the RF‐22, respectively. The median turn‐around time was 6 hr (range 5–7 hr) for NxTAG‐RPP and 12 hr (range 8–16 hr) for RF‐22. The NxTAG‐RPP showed comparable detection rates for most respiratory pathogens, while hands‐on and turn‐around time were considerably shorter. The clinical significance of detecting multiple viruses needs further clinical evaluation. J. Med. Virol. 88:1319–1324, 2016.

Hemolytic uremic syndrome in a 65 year-old male linked to a very unusual type of stx2e and eae harboring O51:H49 Shiga-toxin producing Escherichia coli

ABSTRACT We report on a 65-year-old male patient with a Shiga toxin-producing Escherichia coli O51:H49 gastrointestinal infection and sepsis associated with hemolytic uremic syndrome (HUS) with a fatal outcome. The strains isolated harbored stx 2e and eae, a very unusual and new virulence profile for an HUS-associated enterohemorrhagic E. coli.

In vitro antiallergic effects of aqueous fermented preparations from Citrus and Cydonia fruits.

This study aimed to investigate the immunomodulatory and antiallergic properties of preparations from lemon, Citrus medica L. (citrus), and quince, Cydonia oblonga Mill. (Cydonia), which are used in pharmaceutical products to treat patients suffering from allergic disorders. Preparations were analyzed with respect to their impact on the degranulation capacity from basophilic cells as well as mediator release from activated human mast cells in vitro, including IL-8 and TNF- α secretion. The results show that the degranulation of basophilic cells was diminished only in the presence of Citrus, and this effect was compared to the synthetic drug azelastine. Furthermore, Citrus and Cydonia both inhibited the production of IL-8 and TNF- α from human mast cells, and at low concentrations additive effects were observed. As a positive inhibition control, dexamethasone was used. LC-MS analyses showed that the major phenolic components in extracts from Citrus and Cydonia are eriocitrin and neochlorogenic acid, respectively. Nevertheless, these compounds do not show biological effects at concentration levels detected in their corresponding extracts. In conclusion, the present data provide a rational base for the use of the single pharmaceutical preparations from Citrus and Cydonia in a differentiated treatment of allergic disorders in part by the regulation of soluble allergic mediators from basophilic cells and mast cells.

Safety and effects of two mistletoe preparations on production of Interleukin-6 and other immune parameters - a placebo controlled clinical trial in healthy subjects

BackgroundIn Germany, Iscucin® Populi (IP), a preparation from mistletoe growing on the poplar tree, is used in cancer therapy while Viscum Mali e planta tota (VM), a preparation from mistletoe growing on the apple tree, is used in patients with osteoarthritis. Since mistletoe preparations are suspected to induce production of potentially tumor promoting cytokines like interleukin (IL)-6, further studies on the immunological effects are of interest.MethodsIn this 3-armed randomized, double blind clinical trial healthy volunteers received increasing doses of either IP (strength F, 0.0125%, G, 0.25% and H, 5%, each for 4 weeks), or VM (1:1000 [D3], 1:100 [D2] and 2% each for 4 weeks) or placebo (isotonic solution) subcutaneously twice per week over a period of 12 weeks. Physical examination was performed weekly. Routine laboratory parameters and immunological parameters (C-reactive protein (CRP), differential blood count, lymphocyte subsets, immunoglobulins, IL-6 and tumor necrosis factor (TNF)-α) were analysed every 4 weeks.Results71 subjects were included in the study (IP = 30, VM = 21, placebo = 20) of whom 69 concluded it according to protocol. Application of IP strengths G and H caused strong local reactions at the site of injection. In parallel, a distinct eosinophilia (p < 0.001 compared to placebo) occurred. Furthermore, application of all IP concentrations resulted in an increase of CD4 cell counts (p < 0.05) compared to placebo. Stimulation of IL-6 production, CRP or relevant deviations in other laboratory parameters were not observed. Because of local reactions, IP strengths G and H were considered less tolerable than placebo. VM 2% was slightly less tolerable than placebo, caused only mild local reactions and an only small increase in eosinophile counts.ConclusionTreatment with IP results in eosinophilia and an increase of CD4 cells but not in an increase of IL-6 or CRP. No safety concerns regarding the two mistletoe preparations have been raised by this study. EudraCT-Number 2007-002166-35.Trial registrationClinicalTrials.gov: NCT01378702

Comparison of a UL111a real-time PCR and pp65 antigenemia for the detection of cytomegalovirus

Surveillance of cytomegalovirus (CMV) replication in transplant patients is crucial for the success of transplantation. To compare a CMV pp65 antigenemia (pp65Ag) and a quantitative real‐time PCR targeting the CMV‐UL111a (UL111aPCR), all whole blood samples taken between July 2008 and October 2009 were identified which had been analyzed prospectively by both assays in parallel. Discordant results were re‐analyzed using a published CMV duplex PCR targeting regions UL55 and UL123exon4. Of 720 samples from 81 transplant patients, CMV replication was detected in 244 specimens (34%) by the UL111aPCR (median, 1,019 geq/ml), compared to 113 (16%) detected by the pp65Ag (median, 2/250,000 leukocytes). Concordant UL111aPCR/pp65Ag results were obtained in 561 (78%) samples, being positive in 99 (14%), and negative in 462 (64%). As a rule of thumb, 1 pp65Ag‐positive cell per 250,000 leukocytes corresponded to 1,000 geq/ml CMV DNA of whole blood. Discordant results were found in 159 samples (22%), being UL111aPCR‐positive/pp65Ag‐negative in 145 (91%; median, 650 geq/ml), or UL111aPCR‐negative/pp65Ag‐positive in 14 (9%; median, 1/250,000 cells). Using the duplex PCR targeting the CMV UL55 and the UL123‐exon4 genes, 131 of 139 (94%) discordant UL111aPCR‐positives (median UL111aPCR, 639 geq/ml; median UL55PCR, 715 geq/ml; median UL123PCR, 1,103 geq/ml) were confirmed. Of 14 discordant pp65Ag‐positives, duplex PCR was also negative in 8, and of low copy number in 6. Thus, CMV UL111aPCR provides more sensitive quantitation of CMV replication than pp65Ag, however, discordant results can occur at very low viral loads. J. Med. Virol. 83:2143–2150, 2011.

Constituents from oak bark (Quercus robur L.) inhibit degranulation and allergic mediator release from basophils and mast cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE Oak bark has been used since ancient times in Europaen ethnomedicine because of its adstringent, antimicrobial and hemostatic features, e.g. as a remedy for the treatment of wounds and skin diseases. PURPOSE Oak bark tannins are considered as bioactive natural products, interacting with surface proteins of mucous membranes and might be beneficial for the treatment of allergic diseases. This study investigated the effect of an oak bark decoction (OBD) and isolated tannin fractions on the degranulation capacity and cytokine/chemokine release from rat basophilic cells and human mast cells in vitro, which are essential for the initiation of early- and late-phase allergic reactions. METHODS AND METHODS By chromatographic separation on Sephadex® LH-20 high- and low-molecular weight tannins were separated from OBD and the tannin composition analyzed by HPLC(DAD)-MSn. Then, the OBD and its fractions were tested in degranulation (β-hexosaminidase activity) of allergen-specific-activated basophilic cells in a photometric assay. RESULTS The OBD and the high-molecular tannin fraction showed a dose-dependent inhibition of cell degranulation. Furthermore, the OBD and particularly its high molecular weight tannin fraction exhibited an inhibitory activity on the IL-8-, IL-6- and TNF-α-secretion from stimulated human mast cells, detected and quantified by ELISA. CONCLUSION The OBD and its high-molecular weight tannins revealed an impact on allergic mediator release of basophilic cells and human mast cells and thereby provide a rationale for the topical treatment with OBD preparations.

Safety and immunological effects of Iscucin® Populi and Viscum Mali—A placebo-controlled study

Background Mistletoe preparations Iscucin ® Populi (IP) and Viscum Mali (VM, both WALA GmbH) are licensed in Germany as supportive cancer medication within the concept of Anthroposophical Medicine. Safety and immunological effects in humans of these preparations were for the first time investigated in this study. Methods A 3-armed randomized study in healthy volunteers was performed. The probands injected in increasing doses either IP (strength F, G and H, each for 4 weeks) or VM (strength D3, D2 and 2% each for 4 weeks) or placebo (isotonic solution) subcutaneously twice weekly for a total of 12 weeks. Clinical and safety controls were performed weekly. Immunological outcome parameters (differential blood count, lymphocyte differentiation, interleukin (IL)-6) were analysed every 4 weeks. Results A total of 71 probands were included (IP=30, VM=21, placebo=20), 69 finished the study regularly and were analysed. Application of IP strength G and H caused strong local reactions at the site of injection. In parallel they resulted in distinct eosinophilia ( p p Discussion and conclusion Eosinophilia as an effect of mistletoe-lectin (ML) containing mistletoe preparations is well known and was pronounced due to the high content of ML in IP strength G and H. Most of the probands did not tolerate the full dose of IP strength G and H due to local reactions. Repeated injections induced, however, a certain tolerance (smaller local reactions, less eosinophilia despite inreasing dosages), which was accompanied by induction of mistletoe lectin antibodies in all subjects receiving IP (data not shown). Therefore, IP strength G and H should in general be used only after pretreatment with lower dosages. For the first time there was an increase of T-helper cells during application of mistletoe preparations in a placebo-controlled study. This could be beneficial for the treatment of immunosuppressed cancer patients. Eosinophils play a role in host response against cancer and eosinophilia correlates with a better prognosis in a variety of cancer types. Whether eosinophilia during treatment with IP has a clinical benefit for cancer patients has to be further investigated. Both mistletoe preparations have been shown to be safe. VM induces only mild local reactions and is used as mild counter stimulant in patients with painful osteoarthritis.