Can ELISPOT Be Applied to A Clinical Setting as A Diagnostic Utility for Neuroborreliosis?

The aim of this prospective study was to investigate the diagnostic performance of Borrelia (Bb)-induced interferon (IFN)-γ secretion detected by ELISPOT modified to be feasible for clinical laboratories as a supplementary test to the laboratory diagnosis of Lyme neuroborreliosis (LNB) in an endemic setting. Between 2002 and 2004, patients with symptoms of suspected clinical LNB were included in a study conducted on the Åland islands in the Finnish archipelago, which is a hyper-endemic area for Lyme borreliosis (LB). Fourteen patients with confirmed LNB and 103 patients with non-LNB were included, and the numbers of spontaneous and Bb-induced IFN-γ-secreting cells were assayed by the ELISPOT test. The ELISPOT assay showed a weak diagnostic performance with a sensitivity of 36% and a specificity of 82%. The findings in this study show that this ELISPOT-assay modified to be feasible in clinical routine laboratories is not useful as a supplementary diagnostic tool in the laboratory diagnosis of patients with clinically suspected LNB.

Spontaneously draining acute otitis media in children: an observational study of clinical findings, microbiology and clinical course.

OBJECTIVES: To study the outcome of acute otitis media (AOM) with otorrhoea in children managed initially without antibiotics, in relation to bacterial and clinical findings, and to identify those who may benefit from antibiotics. METHODS: Otherwise healthy, not otitis prone children aged 2-16 y, presenting with AOM with spontaneous otorrhoea, were recruited from primary care and followed at selected ear, nose and throat (ENT) clinics. Specimens for bacterial investigations were obtained; symptoms were registered on a daily basis. The main outcomes measured were the frequency of children treated with antibiotics due to persisting AOM within 9 days in relation to clinical and bacteriological findings, and new AOM within 3 months. RESULTS: Twelve of 71 children who completed the trial received antibiotics during the first 9 days due to lack of improvement. One received antibiotics after 16 days due to relapsing AOM and 6 received antibiotics after 30 days due to new AOM. At 2-4 days following inclusion, over 70% of children showed normalized eardrum status and markedly reduced secretion. Alloiococcus otitidis was found in 23 samples, Streptococcus pneumoniae in 12, Streptococcus pyogenes in 6, and Fusobacterium nucleatum in 5. Mycoplasma pneumoniae, Chlamydia pneumoniae, and Fusobacterium necrophorum were not detected. Antibiotics were prescribed more extensively to children with a pulsating eardrum and abundant purulent secretion. All children with S. pyogenes received antibiotics, whereas children with only A. otitidis did not. CONCLUSIONS: The results suggest that antibiotics are indicated in AOM with otorrhoea and the presence of abundant purulent secretion, a pulsating eardrum, or the presence of S. pyogenes. The presence of only A. otitidis was not associated with a more prolonged course or the need for antibiotics.

Diagnostic performance of cerebrospinal fluid chemokine CXCL13 and antibodies to the C6-peptide in Lyme neuroborreliosis.

OBJECTIVES: The aim of this study was to evaluate the chemokine CXCL13 and C6 antibodies separately and in combination in paired serum/cerebrospinal fluid (CSF) samples in the laboratory diagnosis of Lyme neuroborreliosis (LNB). METHODS: A large retrospective material with paired serum/CSF samples from 261 patients with clinically suspected LNB was investigated. Patients were divided into three main diagnostic groups based on original results of CSF pleocytosis and intrathecal anti-borrelia antibodies (purified flagellum). Levels of CXCL13, albumin, total IgM and IgG in paired samples and C6 antibodies in CSF were compared across diagnostic groups. RESULTS: A sensitivity of 99% and a specificity of 96% were achieved for CSF-Serum CXCL13 ratio. CSF-C6 antibodies performed with a sensitivity of 99% and a specificity of 88.0%. A combination of CSF-Serum CXCL13 ratio and CSF-C6 antibodies, evaluated in parallel, revealed a sensitivity of 99% and specificity of 98%. CONCLUSIONS: This study confirms CSF-CXCL13 as a reliable marker of LNB and suggests improved diagnostic performance especially in children with possible LNB.

Mapping of hormones and cortisol responses in patients after Lyme neuroborreliosis.

BACKGROUND: Persistent symptoms after treatment for neuroborreliosis are common for reasons mainly unknown. These symptoms are often unspecific and could be caused by dysfunctions in endocrine systems, an issue that has not been previously addressed systematically. We therefore mapped hormone levels in patients with previous confirmed Lyme neuroborreliosis of different outcomes and compared them with a healthy control group. METHODS: Twenty patients of a retrospective cohort of patients treated for definite Lyme neuroborreliosis were recruited 2.3 to 3.7 years (median 2.7) after diagnosis, together with 23 healthy controls. Lyme neuroborreliosis patients were stratified into two groups according to a symptom/sign score. All participants underwent anthropometric and physiological investigation as well as an extensive biochemical endocrine investigation including a short high-dose adrenocorticotropic hormone stimulation (Synacthen) test. In addition to hormonal status, we also examined electrolytes, 25-hydroxy-vitamin D and interleukin-6. RESULTS: Eight patients (40%) had pronounced symptoms 2-3 years after treatment. This group had a higher cortisol response to synacthen as compared with both controls and the Lyme neuroborreliosis patients without remaining symptoms (p < 0.001 for both comparisons). No other significant differences in the various baseline biochemical parameters, anthropometric or physiological data could be detected across groups. CONCLUSIONS: Apart from a positive association between the occurrence of long-lasting complaints after Lyme neuroborreliosis and cortisol response to synacthen, no corticotropic insufficiency or other serious hormonal dysfunction was found to be associated with remaining symptoms after treatment for Lyme neuroborreliosis.

Globally disseminated human pathogenic Escherichia coli of O25b-ST131 clone, harbouring blaCTX-M-15 , found in Glaucous-winged gull at remote Commander Islands, Russia.

With focus on environmental dissemination of antibiotic resistance among clinically relevant bacteria, such as the rising ESBL type of resistance among Escherichia coli, we investigated antibiotic resistance levels in wild birds in the Commander Islands and Kamchatka, Russia. Despite overall low resistance levels in randomly selected E. coli (one from each sample), we found multi-resistant ESBL-producing E. coli harbouring blaCTX-M-14 and blaCTX-M-15 using selective screening. Among these multi-resistant ESBL-producing E. coli we found one blaCTX-M-15 harbouring strain belonging to the O25b-ST131 clone, recognized for its clonal disseminated worldwide as a human pathogen. The potential in acquiring resistant bacteria of human origin, especially highly pathogenic clones, as well as downstream consequences of that, should not be underestimated but further investigated.

A novel Chlamydiaceae-like bacterium found in faecal specimens from sea birds from the Bering Sea.

The family Chlamydiaceae contains several bacterial pathogens of important human and veterinary medical concern, such as Chlamydia trachomatis and Chlamydophila psittaci. Within the order Chlamydiales there are also an increasing number of chlamydia-like bacteria whose biodiversity, host range and environmental spread seem to have been largely underestimated, and which are currently being investigated for their potential medical relevance. In this study we present 16S rRNA, rnpB and ompA gene sequence data congruently indicating a novel chlamydia-like bacterium found in faecal specimens from opportunistic fish-eating sea birds, belonging to the Laridae and Alcidae families, from the Bering Sea. This novel bacterium appears to be closer to the Chlamydiaceae than other chlamydia-like bacteria and is most likely a novel genus within the Chlamydiaceae family.

Complex population structure of Lyme borreliosis group spirochete Borrelia garinii in subarctic Eurasia.

Borrelia garinii, a causative agent of Lyme borreliosis in Europe and Asia, is naturally maintained in marine and terrestrial enzootic cycles, which primarily involve birds, including seabirds and migratory passerines. These bird groups associate with, correspondingly, Ixodes uriae and Ixodes ricinus ticks, of which the latter species may bite and transmit the infection to humans. Studies of the overlap between these two natural cycles of B. garinii have been limited, in part due to the absence of representative collections of this spirochete's samples, as well as of the lack of reliable measure of the genetic heterogeneity of its strains. As a prerequisite for understanding the epidemiological correlates of the complex maintenance of B. garinii, the present study sought to assess the diversity and phylogenetic relationships of this species' strains from its natural hosts and patients with Lyme borreliosis from subarctic Eurasia. We used sequence typing of the partial rrs-rrl intergenic spacer (IGS) of archived and prospective samples of B. garinii from I. uriae ticks collected predominantly on Commander Islands in North Pacific, as well as on the islands in northern Sweden and arctic Norway. We also typed B. garinii samples from patients with Lyme borreliosis and I. ricinus ticks infesting migratory birds in southern Sweden, or found questing in selected sites on the islands in the Baltic Sea and Lithuania. Fifty-two (68%) of 77 B. garinii samples representing wide geographical range and associated with I. ricinus and infection of humans contributed 12 (60%) of total 20 identified IGS variants. In contrast, the remaining 25 (32%) samples recovered from I. uriae ticks from a few islands accounted for as many as 10 (50%) IGS types, suggesting greater local diversity of B. garinii maintained by seabirds and their ticks. Two IGS variants of the spirochete in common for both tick species were found in I. ricinus larvae from migratory birds, an indication that B. garinii strains are exchanged between different ecological niches. Notably, B. garinii variants associated with I. uriae ticks were found in each of the six clusters, representing two phylogenetic lineages of this species identified among the studied samples. Our findings suggest that B. garinii in subarctic Eurasia comprises two partially overlapping populations with different levels of genetic heterogeneity, presumably, due to distinctive selective pressures on the spirochete in its marine and terrestrial enzootic cycles.

Antibody responses to borrelia IR(6) peptide variants and the C6 peptide in Swedish patients with erythema migrans.

The aim of this study was to evaluate the antibody responses to different VlsE protein IR(6) peptide variants and the synthetic C6 peptide in acute and convalescent (2-3 and 6 months) serum samples from Swedish patients with clinical erythema migrans (EM). Serum samples were prospectively collected from 148 patients with EM and compared to serum samples obtained from 200 healthy blood donors. The IgG responses to 3 IR(6) peptide variants originating from Borrelia burgdorferi (B. burgdorferi) sensu stricto, B. garinii, and B. afzelii were measured by enzyme-linked immunosorbent assays (ELISAs) and compared to a commercial C6 peptide ELISA. Seropositivity rate in the IR(6) or C6 peptide ELISAs ranged from 32% to 58% at presentation, 30-52% after 2-3 months, and 20-36% after 6 months. At presentation, positive antibodies in any of the 4 ELISAs were found in 66%. In 7/52 (13%), C6-negative EM cases, serological reaction was found to the B. burgdorferi sensu stricto-derived IR(6) peptide. In patients reporting previous LB compared to those without previous LB, significantly higher seropositivity rates were noted for all IR(6) peptides, but not for the C6 peptide. In the serology of EM in Europe, C6 ELISA does not seem to cover all cases. An ELISA using a mixture of B. burgdorferi sensu stricto IR(6) peptide and the C6 peptide could be of value in the serodiagnosis of LB in Europe. Further studies on combinations of variant IR(6) peptides and the C6 peptide in other manifestations of LB are needed to address this issue.

C6-peptide serology as diagnostic tool in neuroborreliosis.

The aim of this study was to evaluate the usefulness of borrelia serology (Quick ELISA C6 Borrelia assay kit) as a diagnostic tool in cases of suspected neuroborreliosis. A retrospective patient material consisting of 124 paired serum and cerebrospinal fluid samples with a positive anti-borrelia antibody index (AI) using the IDEIA Lyme Neuroborreliosis test was compared with 124 AI-negative matched control subjects. The patients were divided into four groups based on presence of pleocytosis and age above or below 12 years. The presence of positive C6 serology in AI-positive patients with pleocytosis was 89% (83/93), significantly different (p<0.01) from in patients without pleocytosis (58%, 18/31). In AI-positive patients aged > or =12 years with pleocytosis, 94% (51/54) had a positive C6 serology. Of AI-positive patients with a symptom duration of more than 30 days, 93% (27/29) were positive by the C6 test. We conclude that the C6 serum test, together with clinical evaluation, is a powerful diagnostic tool in adult (> or =12 years) European patients with suspected neuroborreliosis with a symptom duration of more than 30 days. Patients with suspected neuroborreliosis and positive C6 results should be further investigated by lumbar puncture for definite diagnosis.

Evaluation of phenoxymethylpenicillin treatment of acute otitis media in children aged 2-16.

OBJECTIVE: To study the clinical recovery from acute otitis media (AOM) in children, 2-16 years of age, managed with or without treatment with phenoxymethylpenicillin (PcV). DESIGN: An open, prospective randomized trial. Children aged between 2 and 16 years, presenting with one- or double-sided AOM (without perforation) with symptom duration of less than four days, were included. The children were randomized to PcV for five days or to no primary antibiotic treatment. A health score and compliance were registered on a daily basis for seven days. SETTING: A total of 32 health centres and 72 GPs in south-east Sweden. Subjects. Children aged 2-16 presenting with earache. MAIN OUTCOME MEASURES: Recovery time, symptom duration, frequency of complications (up to three months) and consumption of healthcare services independent of treatment with or without antibiotics. RESULTS: A total of 179 patients carried out the trial; 92 were randomized to PcV, 87 to no primary antibiotic treatment. The median recovery time was four days in both groups. Patients who received PcV had less pain (p <0.001) and used fewer analgesics. There were no significant differences in the number of middle-ear effusions or perforations at the final control after three months. Children randomized to PcV treatment consulted less (p <0.001) during the first seven days. CONCLUSIONS: Our investigation supports that PcV treatment of AOM does not affect the recovery time or complication rates. PcV provided some symptomatic benefit in the treatment of AOM in otherwise healthy children, aged 2-16 years.

Human granulocytic ehrlichiosis as a common cause of tick-associated fever in Southeast Sweden: report from a prospective clinical study.

Between May and December 1998, tick-associated febrile illness was prospectively studied in Southeast Sweden in order to assess the occurrence of human granulocytic ehrlichiosis (HGE). Inclusion criteria were fever (> or = 38.0 degrees C), with or without headache, myalgia or arthralgia in patients with an observed tick bite or tick exposure within 1 month prior to onset of symptoms. Patients with clinical signs of Lyme borreliosis were included. Of the 27 patients included, we identified 4 cases of HGE. Three of the patients had coinfection with Lyme borreliosis, which presented as erythema migrans. All 27 patients presented with a 2-5 d history of fever. None of the clinical signs or laboratory parameters monitored was helpful in predicting ehrlichiosis in this group with tick-associated fever conditions. Within the HGE-negative group (n = 23), 12 patients had clinical or laboratory signs of Lyme borreliosis. For 11 patients, the aetiology of the fever remained unclear. Our results suggest that HGE is common in tick-infested areas of Southeast Sweden, and may occur as a coinfection of Lyme borreliosis. Granulocytic ehrlichiosis should be suspected in patients who present with tick-associated fever, with or without erythema migrans. Ehrlichia serology and PCR should be employed to confirm the diagnosis.

Isolation and characterization of two European strains of Ehrlichia phagocytophila of equine origin.

We report the isolation and partial genetic characterization of two equine strains of granulocytic Ehrlichia of the genogroup Ehrlichia phagocytophila. Frozen whole-blood samples from two Swedish horses with laboratory-verified granulocytic ehrlichiosis were inoculated into HL-60 cell cultures. Granulocytic Ehrlichia was isolated and propagated from both horses. DNA extracts from the respective strains were amplified by PCR using primers directed towards the 16S rRNA gene, the groESL heat shock operon gene, and the ank gene. The amplified gene fragments were sequenced and compared to known sequences in the GenBank database. With respect to the 16S rRNA gene, the groESL gene, and the ank gene, the DNA sequences of the two equine Ehrlichia isolates were identical to sequences found in isolates from clinical cases of granulocytic ehrlichiosis in humans and domestic animals in Sweden. However, compared to amplified DNA from an American Ehrlichia strain of the E. phagocytophila genogroup, differences were found in the groESL gene and ank gene sequences.