Short-course subcutaneous treatment with PQ Grass strongly improves symptom and medication scores in grass allergy.

BACKGROUND: A modified grass allergen subcutaneous immunotherapy (SCIT) product with MicroCrystalline Tyrosine and monophosphoryl lipid-A as an adjuvant system (Grass MATA MPL [PQ Grass]) is being developed as short-course treatment of grass-pollen allergic rhinitis (SAR) and/or rhinoconjunctivitis. We sought to evaluate the combined symptom and medication score (CSMS) of the optimized cumulative dose of 27,600 standardized units (SU) PQ Grass in a field setting prior to embarking on a pivotal Phase III trial. METHODS: In this exploratory, randomized, double-blind, placebo-controlled trial subjects were enrolled across 14 sites (Germany and the United States of America). Six pre-seasonal subcutaneous injections of PQ Grass (using conventional or extended regimens) or placebo were administered to 119 subjects (aged 18-65 years) with moderate-to-severe SAR with or without asthma that was well-controlled. The primary efficacy endpoint was CSMS during peak grass pollen season (GPS). Secondary endpoints included Rhinoconjunctivitis Quality of Life Questionnaire standardized (RQLQ-S) and allergen-specific IgG4 response. RESULTS: The mean CSMS compared to placebo was 33.1% (p = .0325) and 39.5% (p = .0112) for the conventional and extended regimens, respectively. An increase in IgG4 was shown for both regimens (p < .01) as well as an improvement in total RQLQ-S for the extended regimen (mean change -0.72, p = .02). Both regimens were well-tolerated. CONCLUSIONS: This trial demonstrated a clinically relevant and statistically significant efficacy response to PQ Grass. Unprecedented effect sizes were reached for grass allergy of up to ≈40% compared to placebo for CSMS after only six PQ Grass injections. Both PQ Grass regimens were considered equally safe and well-tolerated. Based on enhanced efficacy profile extended regime will be progressed to the pivotal Phase III trial.

Major Allergen Content in Allergen Immunotherapy Products: The Limited Value of Numbers.

The prevalence of allergic disorders has increased drastically over the last 50 years to the extent that they can be considered epidemic. At present, allergen-specific immunotherapy (AIT) is the only therapy that targets the underlying cause of allergic disorders, and evidence of its superiority is based on data accumulated from clinical trials and observational studies demonstrating efficacy and safety. However, several aspects remain unresolved, such as harmonization and standardization of manufacturing and quantification procedures across manufacturers, homogeneous reporting of strength, and the establishment of international reference standards for many allergens. This article discusses issues related to the measurement of major allergen content in AIT extracts, raising the question of whether comparison of products from different manufacturers is an appropriate basis for selecting a specific AIT product. Allergen standardization in immunotherapy products is critical for ensuring quality and, thereby, safety and efficacy. However, lack of harmonization in manufacturing processes, allergen quantification (methodologies and references), national regulatory differences, clinical practice, and labeling shows that the comparison of AIT products based solely on major allergen amounts is not rational and, in fact, impossible. Moreover, when rating the information given for a specific product, it is necessary to take into account further inherent characteristics of products and their application in clinical practice, such as the state of extract modification, addition of adjuvant or adjuvant system, route of administration (sublingual/ subcutaneous), and cumulative dose as per posology (including the volume per administration). Finally, only convincing clinical data can serve as the basis for product-specific evaluation and cross-product comparability of individual products.

Strong dose response after immunotherapy with PQ grass using conjunctival provocation testing.

BACKGROUND: Pollinex Quattro Grass (PQ Grass) is an effective, well-tolerated, short pre-seasonal subcutaneous immunotherapy to treat seasonal allergic rhinoconjunctivitis (SAR) due to grass pollen. In this Phase II study, 4 cumulative doses of PQ Grass and placebo were evaluated to determine its optimal cumulative dose. METHODS: Patients with grass pollen-induced SAR were randomised to either a cumulative dose of PQ Grass (5100, 14400, 27600 and 35600 SU) or placebo, administered as 6 weekly subcutaneous injections over 31-41 days (EudraCT number 2017-000333-31). Standardized conjunctival provocation tests (CPT) using grass pollen allergen extract were performed at screening, baseline and post-treatment to determine the total symptom score (TSS) assessed approximately 4 weeks after dosing. Three models were pre-defined (Emax, logistic, and linear in log-dose model) to evaluate a dose response relationship. RESULTS: In total, 95.5% of the 447 randomized patients received all 6 injections. A highly statistically significant (p < 0.0001), monotonic dose response was observed for all three pre-specified models. All treatment groups showed a statistically significant decrease from baseline in TSS compared to placebo, with the largest decrease observed after 27600 SU (p < 0.0001). The full course of 6 injections was completed by 95.5% of patients. Treatment-emergent adverse events were similar across PQ Grass groups, and mostly mild and transient in nature. CONCLUSIONS: PQ Grass demonstrated a strong curvilinear dose response in TSS following CPT without compromising its safety profile.

Randomized controlled trials define shape of dose response for Pollinex Quattro Birch allergoid immunotherapy.

BACKGROUND: The Birch Allergoid, Tyrosine Adsorbate, Monophosphoryl Lipid A (POLLINEX® Quattro Plus 1.0 ml Birch 100%) is an effective, well-tolerated short course subcutaneous immunotherapy. We performed 2 phase II studies to determine its optimal cumulative dose. METHODS: The studies were conducted in Germany, Austria and Poland (EudraCT numbers: 2012-004336-28 PQBirch203 and 2015-000984-15 PQBirch204) using a wide range of cumulative doses. In both studies, subjects were administered 6 therapy injections weekly outside the pollen season. Conjunctival Provocation Tests were performed at screening, baseline and 3-4 weeks after completing treatment, to quantify the reduction in Total Symptom Scores (as the primary endpoint) with each cumulative dose. Multiple Comparison Procedure and Modeling analysis was used to test for the dose response, shape of the curve and estimation of the median effective dose (ED50 ), a measure of potency. RESULTS: Statistically significant dose responses (P < .01 & .001) were seen, respectively. The highest cumulative dose in PQBirch204 (27 300 standardized units [SU]) approached a plateau. Potency of the PQBirch was demonstrated by an ED50 2723 SU, just over half the current dose. Prevalence of treatment-emergent adverse events was similar for active doses, most being short-lived and mild. Compliance was over 85% in all groups. CONCLUSION: Increasing the cumulative dose of PQBirch 5.5-fold from 5100 to 27 300 SU achieved an absolute point difference from placebo of 1.91, a relative difference 32.3% and an increase in efficacy of 50%, without compromising safety. The cumulative dose response was confirmed to be curvilinear in shape.

Ultra-short-course booster is effective in recurrent grass pollen-induced allergic rhinoconjunctivitis.

BACKGROUND: A relevant proportion of allergic rhinoconjunctivitis (ARC) patients experience recurrent symptoms after successfully completing allergen immunotherapy (AIT). This prospective, controlled, noninterventional study used internationally standardized instruments to determine the clinical effects of a preseasonal, ultra-short-course booster AIT on clinical outcome parameters. METHODS: This two-arm study included patients aged ≥12 years with recurrent grass pollen-induced seasonal AR who had completed a successful course of any grass pollen AIT at least 5 years before enrolment. Overall, 56 patients received one preseasonal short-course booster AIT using tyrosine-absorbed grass pollen allergoids containing the adjuvant monophosphoryl lipid A (MPL® ); 51 control patients received symptomatic medication. The combined symptom and medication score (CSMS) was recorded in the (peak) grass pollen season. Furthermore, concomitant (antiallergic) medication use, the patients' state of health, Mini Rhinoconjunctivitis Quality of Life Questionnaire (MiniRQLQ) results and safety/tolerability of the treatment were assessed. RESULTS: The CSMS in the peak grass pollen season was significantly lower in the booster AIT group (Δ=38.4%, P<.01). Moreover, significantly more patients in this group used no concomitant antiallergic medication throughout the peak grass pollen season. Twice as many patients in the booster AIT group as in the control group reported having a better state of health than in the preceding season. MiniRQLQ results showed significant differences favouring the booster AIT. The booster AIT was generally well tolerated, with only two patients reporting mild, grade 1 systemic adverse events. CONCLUSION: Booster AIT using tyrosine-absorbed allergoids containing the adjuvant MPL® effectively prevents re-occurrence of symptoms in patients with grass pollen-induced ARC.

In vitro evidence for efficacy in food intolerance for the multispecies probiotic formulation Ecologic® Tolerance (Syngut™).

The beneficial effects of probiotics are currently the subject of extensive studies in health and medical research. The aim of this research was to specifically design a new probiotic formulation for supplementation in people suffering from food intolerance. The selection of strains was focussed on the capacity to influence mechanisms of action that are important in development of food intolerance with the following parameters measure: in vitro capacity to produce ß-galactosidase, in vitro strengthening of the epithelial barrier, in vitro stimulation of cytokines produced by regulatory T cells, in addition to assessing fundamental quality criteria (stability, gastrointestinal (GI)-survival, multispecies concept, allergen-free). Ecologic®Tolerance/Syngut™ was subsequently developed consisting of a multispecies concept using 4 different probiotic strains (Bifidobacterium lactis W51, Lactobacillus acidophilus W22, Lactobacillus plantarum W21 and Lactococcus lactis W19). Each of these strains demonstrated ability to survive the GI-tract and strain specific effects in producing ß-galactosidase, strengthening the gut barrier function after immunological-induced stress and inhibiting Th2 cytokines (IL-4, IL-5 and IL-13 (≥50%), in addition to stimulating interleukin-10 levels; thus, providing in vitro evidence for the efficacy of the selected strains to provide beneficial effects in patients suffering from food intolerance.

Non-allergic, mastocytosis-associated rhinitis.

BACKGROUND: Systemic mastocytosis (SM) is a clonal proliferative disorder of mast cells (MC) that causes pathological accumulation of mast cells in various tissues, which results in clinical symptoms (e.g. diarrhoea, urticaria) due to MC mediator release. Previous studies have shown that up to fifty percent of rhinitis symptoms in SM patients are non-allergic and it has been assumed that these nasal complaints in SM patients are due an increased nasal mast cell burden. Nevertheless, to date there are no data supporting this hypothesis. OBJECTIVE: The study aims to investigate if the presence of allergy-suggesting nasal complaints in non-allergic SM patients is correlated with objective measure of nasal mast cell burden. PATIENTS AND METHODS: Eleven adult patients with systemic mastocytosis underwent a comprehensive rhinologic work-up. All patients fulfilled the clinical ARIA criteria for rhinitis. The allergologic work-up included serological allergy testing, determination of tryptase levels (serum and nasal secretion), skin prick testing, and nasal provocation testing. RESULTS: Ten out of eleven SM patients with clinical persistent allergic rhinitis were found to be non-allergic. In these patients, the most predominant symptoms were rhinorrhea, sneezing, and itching. All three symptoms were strongly correlated with the nasal tryptase level but not to the level of serum tryptase. CONCLUSION AND CLINICAL RELEVANCE: Non-allergic persistent nasal complaints in systemic mastocytosis were significantly correlated with elevated nasal tryptase level as a measure of local MC burden. Furthermore, elevated nasal tryptase correlated with persistent rhinorrhea, sneezing, and itching as predominant symptoms, which seems to characterize a non-allergic mastocytosis-associated rhinitis (NAMAR) in systemic mastocytosis.

[Food allergies: significance, interfaces, and prospects in otorhinolaryngology]. / Nahrungsmittelallergien: Bedeutung, Schnittstellen und Perspektiven der HNO-Heilkunde.

Perceived and objective food hypersensitivity reactions are increasing steadily in the western countries. Therefore, otorhinolaryngologists are facing more frequently the challenge to evaluate individual food hypersensitivity reactions, to set these reactions into context with otorhinolaryngologic symptoms and to start systemic and appropriate diagnostic procedures and possibly therapy. Although a good portion of the perceived food hypersensitivity reactions can neither be objectivated nor causaly linked to the occurring symptoms, the recent literature provides documentation, suggestions and prospects for the link between food hypersensitivity reactions and distinct ENT-diseases. Based on that, this review intends to provide an overview of the current knowledge about the impact of food hypersensitivity reactions on otorhinolaryngologic diseases. The aim of the article is to give support in assessing this intricate issue in the daily otorhinolaryngological practice.

[Eosinophilic esophagitis : a cause of dysphagia]. / Die eosinophile Osophagitis : Eine Differenzialdiagnose bei Dysphagie.

BACKGROUND: Eosinophilic esophagitis (EE) is a chronic, interleukin-5-driven inflammatory disease of the esophagus, causing dysphagia and esophageal food impactions. We analyzed the diagnostic results of patients with suspected or proven EE and in this article discuss the relevant aspects of this disease. PATIENTS AND METHODS: Sixteen patients suffering from dysphagia or recurrent esophageal food impactions underwent rigid esophagoscopy to exclude EE. In six patients, 24-h pH monitoring was performed to exclude laryngopharyngeal reflux (LPR). RESULTS: EE was diagnosed in only one patient, a boy with a history of peanut allergy and recurrent esophageal food impactions. In six patients, histological examination of biopsies revealed reflux esophagitis indicating gastroesophageal reflux disease (GERD). Using 24-h pH monitoring, LPR was diagnosed in four of six patients. CONCLUSIONS: Even in patients presenting with typical symptoms of EE, this disease is rarely found. However, in male patients with asthma, allergies, or a history of recurrent esophageal food impactions, EE must be excluded. The most important differential diagnoses of EE are GERD and LPR.

[Schwannoma of the nasal cavity: a case report]. / Schwannom der Nasenhaupthöhle: Eine Falldarstellung.

Schwannomas of the nasal cavity and paranasal sinuses are quite rare, with 4% occurring in this location. Most of them are benign and do not recur when totally removed by surgery. It is very important to distinguish between schwannoma and primary benign neurofibroma. Neurofibromas are lesions having the possibility for malignant transformation and recurrence. A case of schwannoma in the nasal cavity is reported, and the diagnostic and therapeutic procedures, as well as recommendations from the literature, are described. The histological and immunohistochemical features are discussed in detail to draw a distinction between schwannoma and neurofibroma. In cases of intranasal and paranasal lesions, the existence of a schwannoma must be considered. Differentiating between schwannoma and neurofibroma is important for estimating the risk of malignant transformation and recurrence.

[Polyposis nasi--improvement in quality of life by the influence of leukotrien receptor antagonists]. / Polyposis nasi--Besserung der Lebensqualität durch Leukotrien-Rezeptorantagonisten.

BACKGROUND: Sulfido-Leukotrienes are important inflammatory mediators of bronchial asthma, intolerance of acetylsalicylic acid (ASA), polyposis nasi and allergic rhinitis. Receptorantagonists like Montelukast constitute a well-established asthma- and ASA intolerance-therapy. The aim of our study was to evaluate changes in patients Health-Related-Quality-of-Life (HRQL) during Montelukast-monotherapy of nasal polyposis. METHODS: The study was performed in a prospective, double blind and placebo-controlled matter. The study included 30 patients of our ENT outpatient's dept. (77 % male, mean age 49 yrs), suffering from nasal polyposis grade II to IV. Polyps were endoscopically graded, nasal Eosinophilic Cationic Protein (ECP) was measured, and HRQL-score was taken prior to and four weeks after Montelukast-(0 - 0 - 10 mg) compared to placebo. An established HRQL-questionnaire - including 25 items, summarized in 6 symptom-groups - was used. Given was a symptom-score of 1 (not troubled) to 4 (extremely troubled). RESULTS: Patients treated with Montelukast improved their nasal symptoms (Delta HRQL-score 0.58 +/- 0.94, P < 0.01), practical problems (Delta HRQL-score 0.42 +/- 0.71, P < 0.05), headaches (Delta HRQL-score 0.38 +/- 0.56, P < 0.05), non-nasal symptoms (Delta HRQL-score 0.35 +/- 0.92, P < 0.05), sleep (Delta HRQL-score 0.26 +/- 0.71) and emotional problems (Delta HRQL-score 0.18 +/- 0.75). Intranasal ECP (Delta 210.67 ng/ml +/- 332.68) and polyp grading (Delta 0.72 +/- 1.77) tended to improve as well, but did not reach statistical significance. Patients treated with placebo revealed no significant changes neither in HRQL-score, ECP, nor polyp grading. CONCLUSIONS: Montelukast-therapy of nasal polyposis significantly improved patient's HRQL in 4 out of 6 symptom-groups. Measuring HRQL proofed to constitute a more sensitive tool than looking at eosinophilic parameters of inflammation or polyp size.

[Diagnosis of persistent rhinitis]. / Was steckt hinter dem Dauerschnupfen?

A total of 9923 patients with persistent rhinitis were characterized on the basis of their symptoms and in vitro data. The most bothersome symptom in these patients was nasal obstruction. "Post-nasal drip" proved to be untypical for allergic rhinitis. In all the diseases investigated, involvement of eosinophilic granulocytes and mast cells was found to vary. In addition to endoscopy and imaging procedures, the determination of selective in vivo and in vitro parameters represents an essential part of the diagnostic work-up of persistent rhinitis, which thus proves to be a domain of the allergological rhinologist.

[Lethal end of a group C streptococcal necrotizing fasciitis in a healthy female]. / Letaler Verlauf einer nekrotisierenden Fasziitis mit beta-hämolysierenden Streptokokken der Gruppe C bei einer gesunden Patientin.

The necrotizing fasciitis (NF) is a life threatening, quickly progressive soft-tissue infection characterized by necrosis of the superficial fascia. It is very rare in the head and neck region. Usually this infection attacks weak patients with chronic diseases, such as diabetes or cancer. In adults the most common organisms implicated in craniocervical NF are Streptococcus pyogenes, Staphylococcus aureus, Streptococcus viridans and Peptostreptococci. The typical infectious entrance is a deep wound, after surgery or a peritonsillar abscess. We describe a very unusual case of a craniocervical NF with a deadly outcome in a sixty year old female. The patient had no chronic diseases and went to an ENT doctor with an acute tonsillitis for 3 days. Within the next three hours the general condition became dangerous to life. Therefore she was brought to us for immediate therapy. But despite fast and aggressive surgery of the neck, throat and thorax the patient died of a toxic schock syndrome on the intensive care unit within a few hours. The microbiology showed just group C Streptococcus. The postmortem examination confirmed the NF with starting point in the left tonsil after acute tonsillitis. Such a foudroyant course of a craniocervical NF with group C streptococci in a healthy patient is not published yet.

[Clinical symptoms and mediators in the allergic early and late phase reaction]. / Klinik und Mediatorfreisetzung in der allergischen Früh- und Spätphasenreaktion.

BACKGROUND: This examination focused on the allergic early and late phase reaction via nasal symptom scores, acoustic rhinometry, and the determination of mediators possibly involved in late phase eosinophilia. We examined nasal secretions for IL-5; the chemokines IL-8, MCP-1, and Eotaxin; the adhesion molecule sVCAM-1, and the leukotriene LTC4 for their suggested impacts on tissue eosinophilia. METHODS: 13 patients suffering from seasonal allergic rhinitis were challenged intranasally out of the natural pollen season by their specific allergen. In a time window of 8 h following the provocation, patients completed symptom questionnaires, and underwent acoustic rhinometry. Nasal secretions were gained by the cotton wool method over a time period of 8 h. Nasal secretions were analyzed for the above mentioned mediators. RESULTS: Individual evaluation of the acoustic rhinometry measurements revealed an early phase reaction in 100 % of the cases and a late phase reaction in 92 %. The need to sneeze and a runny nose were the strongest symptoms during the allergic early and late phase reaction. A typical late phase kinetic was observed for IL-5, MCP-1, Eotaxin, sVCAM-1, and LTC4. IL-8 was characteristic for early phase reaction but increased in late phase as well. CONCLUSIONS: The need to sneeze, a runny nose, and the overall quality of life were most apt to evaluate the allergic early and late phase reaction. Highly significant correlations between nasal obstruction and acoustic rhinometry measurements indicate a high sensitivity of visual analogue scales in the representation of minimal changes in nasal symptom scores during the allergic reaction. Our data point to a relevant role of the TH2 cytokine IL-5; of the chemokines IL-8, MCP-1, and Eotaxin; of the adhesion molecule sVCAM-1, and of the leukotriene LTC4 for the allergic late phase eosinophilia.

[Susac's syndrome. A rare microangiopathy of cochlea, retina, and brain]. / Susac-Syndrom. Eine sehr seltene Mikroangiopathie von Kochlea, Retina und Gehirn.

Susac's syndrome is a rare disease characterized by encephalopathy, retinal artery occlusion, and a sensorineural hearing loss. Diagnosis may be difficult since most specialists are not familiar with this angiopathy. However, the typical symptom complex can mimic different pathologies, therefore requiring the attention of radiologists, ENT specialists, and ophthalmologists. We present a rare case of Susac's syndrome unveiled by audiometry results, MR imaging of the brain, and the ophthalmological findings..

[Humoral mucosal immunity in allergic rhinitis]. / Humorale Mukosa-Immunantwort bei der Rhinitis allergica.

BACKGROUND: Mucosa-immunologic aspects are gaining an increasing awareness in the pathophysiology of type I allergies. Humoral mucosal immune responses are dominated by secretory IgA, but there is evidence for a relevant role of IgG in nasal mucosa-associated lymphoid tissue. OBJECTIVE: was to measure allergen-specific immunoglobulins (IgA and IgG) in nasal secretions as an expression of a humoral mucosal immune response in allergic rhinitis. For tissue eosinophilia we studied nasal Eosinophilic Cationic Protein (ECP) and for mast cell activation nasal tryptase. METHODS: Nasal secretions of 40 patients suffering from allergic rhinitis were analyzed for allergen-specific IgA, IgG, and IgE, and for ECP and tryptase. Patients were highly sensitized against the major allergens of house dust mites, timothy, and birch pollen. 43 non-atopic individuals served as controls. In order to study possible effects of the actual pollen season on the studied parameter we secondly compared patients allergic to seasonal allergens co- (n = 28) and extra-seasonally (n = 41). In order to determine a possible influence of allergen-specific IgA in eosinophilic degranulation we additionally studied 5 patients after nasal allergen challenge. RESULTS: In allergic rhinitis we found significantly increased levels of allergen-specific immunoglobulins of all studied subclasses and allergens in nasal secretions. Comparison of nasal ECP and tryptase showed significantly increased concentrations in allergic individuals as well. Co-seasonally we found elevated allergen-specific IgE, ECP, and tryptase but lower concentrations of allergen-specific IgA and IgG. There was no association between late phase eosinophilia and IgA concentrations after local allergen challenge. CONCLUSIONS: The occurrence of allergen-specific immunoglobulins in nasal secretions is interpreted as a local humoral mucosal immune response. The physiologic role of local allergen-specific immunoglobulins is not clear to date. Involvement in degranulation of eosinophils or mast cells, like suggested before, seems unlikely.

In vitro diagnosis of chronic nasal inflammation.

BACKGROUND: Differential diagnosis of chronic nasal inflammation is insufficient when based solely on clinical examination and radiography of paranasal sinuses. Patients complain about more or less similar symptoms. Activation of mast cells and eosinophils is pivotal in nasal inflammation. OBJECTIVE: To compare tryptase and eosinophilic cationic protein (ECP) in nasal secretions in different forms of chronic nasal inflammation and to establish norm values. METHODS: The study included 1710 patients presenting with nasal complaints. Nasal secretions were gained by the cotton wool method and analysed for tryptase, as a marker of mast cell activation, and for ECP, as a marker of tissue eosinophilia and activation. Patients were grouped according to their diagnosis: chronic, non-allergic rhinosinusitis (sinusitis, n=194), non-allergic nasal polyposis (polyposis, n=138), non-allergic rhinitis with eosinophilia syndrome (NARES, n=198), isolated perennial allergic rhinitis (AR) (n=126), isolated seasonal AR (n=132), and patients allergic to both, seasonal and perennial allergens (n=193). Seven hundred and twenty-nine patients with nasal complaints due to a deviated septum and without any nasal inflammation served as controls. RESULTS: Nasal tryptase was highly significantly (P<0.001) elevated in polyposis, NARES, and in AR. ECP was highly significantly (P<0.001) elevated in all groups of patients suffering from chronic nasal inflammation. Based on our data and method we established norm values (95% confidence interval of mean value) for nasal tryptase in healthy adults, ranging from 12.0 to 18.7 ng/mL and for ECP ranging from 84.4 to 102.6 ng/mL. CONCLUSION: Mast cells and eosinophils are involved in non-allergic and allergic forms of chronic nasal inflammation. We established an in vitro assay for tryptase and ECP in nasal secretions and defined norm values based on our data and method. In vitro measurement of biological markers in nasal secretions provides important information for differential diagnosis and therapeutic strategies of chronic nasal inflammation.

Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources.

AIMS: The genus Enterococcus includes opportunistic pathogens such as E. faecalis and E. faecium, and is also used to assess water quality. Speciation of enterococci in environmental studies can be particularly problematic, therefore protocols for unambiguous, DNA-based analysis could receive wide use in applications ranging from water quality monitoring to microbial source tracking. The goal of this work was to investigate the usefulness of PCR for speciation of putative, biochemically identified E. faecalis and E. faecium isolated from water, faeces and sewage. METHODS AND RESULTS: Putative enterococci (n = 139) were isolated on mEI agar from dog, human, gull and cow faeces, and from sewage, freshwaters and marine waters. A total of 128 isolates passed standard physiological tests for the genus, and were speciated by the API 20 Strep (APIStrep) biochemical test system. 42.2% were identified as E. faecalis, and all were confirmed by PCR. 19.5% were biochemically identified as E. faecium, but only seven were PCR-positive. CONCLUSIONS: The 16S rDNA of PCR-positive and PCR-negative E. faecium, including isolates that were inconclusively identified by APIStrep, was sequenced. All formed a monophyletic clade with E. faecium sequences in Genbank. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical identification of E. faecalis agreed 100% with PCR assays, therefore a simple protocol of isolation on mEI followed by PCR should be useful for environmental studies. Discrepancies among biochemical identification, PCR confirmation and DNA sequencing were noted for E. faecium, indicating that routine isolation/identification of E. faecium from environmental samples is a much more difficult task.