Comparison of two commercially available ELISA antibody test kits for detection of human antibodies against Coxiella burnetii.

BACKGROUND: Q fever is a zoonosis caused by Coxiella burnetii. The disease is emerging in many parts of the world, likely in part due to increased awareness and the availability of better diagnostic tests. The clinical diagnosis of Q fever is difficult, and most confirmed cases rely on serology. METHODS: This study compared the sensitivity, specificity, and performance of 2 commercial enzyme-linked immunosorbent assay (ELISA) kits, with a commercial microimmunofluorescence antibody test (IFA) used as reference. RESULTS: One of the ELISA kits showed a higher sensitivity and a lower cross-reactivity than the other kit. Likewise, the same kit was superior when comparing the area under the receiver operating characteristic curves. CONCLUSIONS: The results support the continued use of IFA as a primary serological test for Q fever; for large numbers of samples, an ELISA kit can be used as a screening tool, if followed by a confirmatory IFA test.

Quantiferon test for tuberculosis screening in sarcoidosis patients.

BACKGROUND: Tumour necrosis factor-alpha (TNF-α) inhibitors have been introduced in the treatment of refractory sarcoidosis. These biologics may reactivate latent tuberculosis infection (LTBI). Despite its known limitations, the tuberculin skin test (TST) is currently used for the diagnosis of LTBI in Danish sarcoidosis patients. We report the results of a screening using the interferon-gamma release assay (IGRA) QuantiFERON TB Gold (QFN) for the diagnosis of LTBI. We aimed to assess whether the QFN is reliable for diagnosing LTBI among sarcoidosis patients and if results are influenced by disease activity or immunosuppressive treatment. METHODS: A prospective study was performed from 2005 to 2007 among sarcoidosis patients who were candidates for TNF-α inhibitor treatment. Information on immunosuppressive treatment was obtained from the medical records. Disease activity was assessed by biochemistry, chest roentgenograms and pulmonary function tests. The predictive value of QFN results was evaluated by follow-up in the Danish National Tuberculosis Registry. RESULTS: A total of 44 sarcoidosis patients (22 men) with a median age of 39 y (range 25-59 y) were enrolled; 93% had a negative QFN test result and 7% had an indeterminate result. Forty-three percent had disease activity and 57% (n = 25) received immunosuppressive treatment. There was no significant difference in QFN interferon-γ response between subjects with or without disease activity (p > 0.4) and between treated vs non-treated patients (p > 0.5). At follow-up using the Danish tuberculosis registry, there was no occurrence of tuberculosis among study participants. CONCLUSIONS: The predictive value of the QFN seems good among Danish sarcoidosis patients and the results appear to be unaffected by sarcoidosis disease activity and immunosuppressive treatment.

Is sarcoidosis a rickettsiosis? An archival study.

BACKGROUND: Based on earlier research, Rickettsia helvetica could possibly be involved in the pathogenesis of sarcoidosis. Rickettsiae are transmitted to humans by a tick vector, Ixodes ricinus; this tick is highly prevalent in Northern Europe. We aimed to investigate the association between evidence of rickettsiae and sarcoidosis in histological samples. METHODS: We included formalin-fixed, paraffin-embedded mediastinal lymph node biopsies from 52 ethnic Danish patients with sarcoidosis and compared these with 50 biopsies from ethnic Danish patients with mediastinal lymphadenopathy of other causes. Samples were analysed for: (1) rickettsial DNA by real-time polymerase chain reaction (PCR) and (2) rickettsial rDNA (ribosomal DNA) by a specific fluorescence in situ hybridization technique (FISH). RESULTS: Rickettsia was not detected in biopsies by real-time PCR and/or FISH analyses. CONCLUSION: Our results do not support the hypothesis that Rickettsia is involved in the pathogenesis of sarcoidosis.

Is sarcoidosis a rickettsiosis?

The pathogenesis of sarcoidosis is still largely unknown. The generally accepted theory is that genetically predisposed individuals develop the sarcoid disease reaction as a response to one or more unknown antigen(s). A single study by Nilsson et al has related the development of sarcoidosis to an infection with Rickettsia helvetica. The aim of this thesis was to investigate whether a rickettsial infection is involved in the pathogenesis of sarcoidosis. We used different microbiological methods as serology, polymerase chain reaction, and fluorescent in situ hybridization (FISH) on samples from patients with sarcoidosis and control patients. The thesis compiles the results from four separate studies: The second paper describes a serological survey in historical patient sera. None of the results from the studies supported the hypothesis of Rickettsia being involved in the pathogenesis of sarcoidosis. In conclusion, we could not find evidence to support the primary hypothesis of the study, that a rickettsial infection should be involved in the pathogenesis of sarcoidosis.

Q fever in Greenland.

We report a patient with Q fever endocarditis in a settlement in eastern Greenland (Isortoq, Ammassalik area). Likely animal sources include sled dogs and seals. Q fever may be underdiagnosed in Arctic areas but may also represent an emerging infection.

A novel fluorescent in situ hybridization technique for detection of Rickettsia spp. in archival samples.

A novel, sensitive and specific method for detecting Rickettsia spp. in archival samples is described. The method involves the use of fluorescently marked oligonucleotide probes for in situ hybridization. Specific hybridization of Rickettsia was found without problems of cross-reactions with bacterial species shown to cross-react serologically.

Evaluation of serological tests for the diagnosis of rickettsiosis in Denmark.

Two commercially available immunofluorescence assays (IFA) were compared using historical sera evaluated for rickettsial antibodies by the Weil-Felix test. An IFA test produced by Focus Diagnostics prepared with Rickettsia rickettsii and R. typhi antigens was compared with a custom made kit from Fuller Laboratories with R. rickettsii, R. typhi, R. conorii and R. helvetica as antigens. The serum panel used for the comparison included Weil-Felix-positive and -negative samples. Cross-reactions were analyzed using serum samples from patients with clinical symptoms similar to those of rickettsiosis. When analyzing the data using the manufacturers' cut-off values, 41% of samples from presumably healthy blood donors were found positive for spotted fever group Rickettsia antibodies. This does not correlate to the general picture of rickettsiosis in Denmark. Furthermore, sera with Coxiella burnetii antibodies were found to be cross-reacting in both tests. When applying cut-off values calculated on the 95% percentile on data from blood donor serum samples, there was no significant difference between the two kits. Moreover, when using the newly established cut-off, cross-reactions were eliminated.

A prospective study evaluating the presence of Rickettsia in Danish patients with sarcoidosis.

Rickettsia helvetica has previously been proposed as an aetiological agent in sarcoidosis. The purpose of the present study was to detect possible signs of Rickettsia infection in a Danish population of patients with sarcoidosis. Twenty-six patients with newly diagnosed sarcoidosis were prospectively enrolled in the study. The diagnosis was confirmed by biopsy in 18 and by clinical characteristics in 8 patients; 11 patients with different non-sarcoid lung diseases were recruited as controls. We obtained information regarding tick exposure and sarcoid disease manifestations by a structured interview. Evidence of rickettsial infection was assessed by an immunofluorescence assay testing for antibodies towards Rickettsia as well as specific real-time polymerase chain reaction (PCR) on lung biopsy specimens. We performed fluorescent in situ hybridization (FISH) on the biopsies to detect rickettsial and eubacterial rRNA. One sarcoidosis patient had serum rickettsial IgG antibodies above the chosen cut-off level. We found no positive rickettsial PCR or FISH analyses in any of the biopsy specimens. One sarcoid patient sample and 1 control sample contained unidentified bacteria. There was no difference in the reported frequency of tick bite between patients and controls. In conclusion, we found no evidence of Rickettsia being involved in the pathogenesis of sarcoidosis in Denmark.

Ficolins and Mannose-Binding Lectin in Danish patients with sarcoidosis.

Mannose-Binding Lectin (MBL) is a prognostic marker in pulmonary diseases. Ficolins, sharing many structural and functional similarities with MBL, may also be involved in the pathogenesis of pulmonary diseases. The objectives of the study were to establish whether plasma concentrations of Ficolin-2, -3, and MBL in Danish patients with sarcoidosis and control persons differed and whether they were of prognostic significance. We retrospectively included 46 consecutive patients (26 male, 20 female) and 51 age- and sex-matched healthy control persons (28 male, 23 female). Information about the patients was obtained from their medical records. We measured plasma concentrations of Ficolin-2, -3, and MBL using ELISA. There was a significant difference in the patients' mean Ficolin-3 plasma level (14.9 microg/ml; +/-2SD: 6.7-23.1) compared with the control persons' (21.6 microg/ml; +/-2SD: 12.7-30.5). The difference was 6.7 microg/ml (95% CI: 5.0-8.4 microg/ml; p<0.001). In the patients, Ficolin-3 correlated inversely with the CD4(+)/CD8(+)-ratio (Spearman's Rho=-0.37; p=0.021; n=39). There were no significant differences in plasma concentrations of Ficolin-2 or MBL between the two groups. Ficolin-3 concentrations were lower in plasma from patients with sarcoidosis. This suggests a possible involvement of Ficolin-3 in the complex pathophysiology of sarcoidosis. However, we could not show the applicability of Ficolin plasma level measurement as a marker of disease activity or of prognostic significance in sarcoidosis.

[Treatment of chronic anal fissure with topically applied nitroglycerin ointment. A systematic review of evidence-based results]. / Behandling af kronisk analfissur med lokalt appliceret nitroglycerinsalve. En systematisk gennemgang af evidensbaserede resultateroversigtsartikel.

This study explores the evidence-based background for treating chronic anal fissure with topically applied nitroglycerin (NTG): in part the general effect of NTG and in part how its effect compares to that of surgery, which has been claimed to have long-term complications like incontinence for flatus and faeces. Ten randomised clinical trials published up to July 2001 were retrieved. In five of six studies, NTG had an effect on healing that was better than that of placebo or lignocaine. Headache is a common side effect of the treatment. Lateral internal sphincterotomy, the operation of choice for chronic anal fissure, and topical NTG were compared in four trials. Surgery had a better healing rate, but more late complications. The results suggest that in 31-65% of patients an operation could be avoided with NTG therapy. Topically applied 0.2% nitroglycerin three times a day for four weeks is therefore the primary choice in the treatment of anal fissures. But the possibility still remains that the observed effect of NTG may be the outcome of publication bias.

The effect of liraglutide on endothelial function in patients with type 2 diabetes.

This single-centre, 12-week, double-blind, placebo-controlled trial assessed how the human glucagon-like-peptide 1 analogue liraglutide impacted endothelial function in adult patients (n = 49) with type 2 diabetes and no overt cardiovascular disease. Patients were randomized to liraglutide, placebo or glimepiride. At baseline and Week 12, venous occlusion plethysmography was used to measure forearm blood flow (FBF) in response to acetylcholine (ACh) and sodium nitroprusside (SNP) before and after (L)-N(G)-monomethyl arginine (L-NMMA) infusion. At Week 12, ACh-mediated FBF increased with liraglutide and decreased with placebo; however, the between-treatment difference was not significant (p = 0.055). Inhibition of ACh-mediated FBF after L-NMMA infusion increased with liraglutide and decreased with placebo; this between-treatment difference was also not significant (p = 0.149). No change in FBF was observed with SNP. Liraglutide did not significantly impact endothelium-dependent vasodilation after 12 weeks; however, additional investigations looking at the effect of liraglutide on endothelial function in alternative vasculature and during the postprandial period are warranted.

Liraglutide achieves A1C targets more often than sitagliptin or exenatide when added to metformin in patients with type 2 diabetes and a baseline A1C <8.0%.

OBJECTIVE: Compare the safety and efficacy of liraglutide to that of sitagliptin or exenatide as add-on to metformin in patients with type 2 diabetes (T2D) and glycated hemoglobin (A1C) <8.0%. METHODS: Post hoc analysis of 26-week data from liraglutide 1.8 mg once daily (OD) versus exenatide 10 µg twice daily (LEAD-6) and liraglutide 1.8 mg OD versus sitagliptin 100 mg OD (LIRA-DPP-4); only patients treated as add-on to metformin with baseline A1C <8.0% were included. Efficacy analysis was performed on the intention-to-treat population with missing values imputed by last observation carried forward. RESULTS: More patients achieved A1C targets (<7.0% and ≤6.5%) with liraglutide versus exenatide or sitagliptin; the difference was greatest for A1C ≤6.5% (LEAD-6: 65% versus 35%; odds ratio [OR]=3.37, 95% confidence interval [CI]: 1.31-8.63; P = .01 or LIRA-DPP-4: 53% versus 19%; OR = 4.78, 95% CI 2.10 to 10.87; P = .0002). Significantly more patients achieved a composite endpoint of A1C <7.0% with no weight gain or hypoglycemia with liraglutide compared with exenatide (78% versus 42%; OR = 4.99, 95% CI: 1.77 to 14.04; P = .0023) or sitagliptin (61% versus 21%; OR = 5.95, 95% CI: 2.66 to 13.29; P<.0001). All treatments were well tolerated, there was no major hypoglycemia and few patients (8 to 10%) experienced minor hypoglycemia. CONCLUSION: When added to metformin in patients with an A1C <8.0%, more patients using liraglutide 1.8 mg reached A1C targets than with exenatide or sitagliptin. Sitagliptin had particularly low efficacy in this analysis. These data support the use of liraglutide 1.8 mg as a safe and effective alternative to sitagliptin or exenatide following metformin failure in patients with an A1C <8.0%.

One-year sustained glycemic control and weight reduction in type 2 diabetes after addition of liraglutide to metformin followed by insulin detemir according to HbA1c target.

AIM: To investigate durability of efficacy and safety over 1 year of the sequence of liraglutide added to metformin followed by add-on insulin detemir if glycated hemoglobin (HbA1c) remains ≥7.0%. METHODS: Patients previously uncontrolled on metformin±sulfonylurea with HbA1c ≥7.0% after 12 weeks of adding liraglutide 1.8mg to metformin (run-in; sulfonylurea discontinued) were randomized 1:1 to 52 weeks' open-label add-on detemir (randomized treatment [RT] group; n=162) or continuation without detemir (randomized control [RC] group; n=161). Patients with HbA1c <7.0% continued 52 weeks' unchanged treatment (observational group; n=498). RESULTS: Run-in HbA1c improvement from 8.3% to 7.6% (-0.6%) was further enhanced in the RT group (-0.50%) and maintained in the RC group (+0.01%) over 52 weeks; estimated treatment difference (ETD)[95%CI]: -0.51 [-0.70;-0.31]; P<0.0001. More RT (52%) than RC patients (22%) achieved HbA1c <7.0% at 52 weeks (P<0.0001). Run-in weight loss (-3.5kg) was maintained in the RT (-0.05kg) and enhanced in the RC group (-1.02kg) after 52 weeks; ETD [95%CI]: 0.97 [0.04;1.91]; P=0.04. No major hypoglycemia occurred; minor hypoglycemia rates were low across groups (0.034-0.228 events/patient-year). CONCLUSIONS: Supplementing metformin+liraglutide with detemir for 52 weeks improved glycemic control with sustained weight loss and low hypoglycemia rate.

The BTNL2 A allele variant is frequent in Danish patients with sarcoidosis.

BACKGROUND: The butyrophilin-like 2 (BTNL2) gene is located on chromosome 6p21.3 close to the HLA-class II genes. An association has been reported between sarcoidosis and a single nucleotide polymorphism in BTNL2, rs2076530, also termed the A allele. OBJECTIVES: To evaluate whether patients with sarcoidosis carry the A allele more frequently than healthy subjects. METHODS: The series comprised 87 ethnic Danes with sarcoidosis and 113 healthy control subjects. Analysis of rs2076530 was performed by Taqman assay, polymerase chain reaction and sequencing of genomic DNA. RESULTS: Sarcoidosis patients had a higher frequency of the A allele than controls (73.9% vs 55.8%) (P < 0.025). The frequency of GG, GA and AA genotype was 5.7%, 40.2% and 54.0% in patients vs 16.0%, 56.6% and 27.4% in controls (P < 0.001). The AA genotype was associated with increased risk of sarcoidosis in both a dominant [odds ratio (OR) 3.1; 95% confidence interval (CI) 1.1-8.7; P < 0.03] and a recessive model (OR 3.1; 95% CI 1.72-5.61; P < 0.001). Population attributable fraction for disease was 50% in a dominant model and 25% in a recessive model. CONCLUSIONS: The BTNL2 A allele variant occurs with a high frequency in Danish patients with sarcoidosis and the AA genotype is associated with a ∼threefold higher risk of sarcoidosis than the GG genotype. Our results should encourage future studies on the interrelationship between the BTNL2 protein and granuloma formation in sarcoidosis.

The continuing search for Mycobacterium tuberculosis involvement in sarcoidosis: a study on archival biopsy specimens.

INTRODUCTION: Increasing evidence indicates that mycobacteria may be involved in the aetiology and pathophysiology of sarcoidosis. OBJECTIVES: To investigate the association between Mycobacterium tuberculosis complex infection and sarcoidosis. METHODS: Mediastinal lymph node biopsy specimens (formalin-fixed, paraffin-embedded) from 52 Danish patients with sarcoidosis, 50 patients with mediastinal lymphadenopathy of other non-mycobacterial causes (negative controls) and 12 patients with histologically and/or culture-verified mycobacteriosis (positive controls) were included in the study. Biopsy samples were analysed for the presence of Mycobacterium tuberculosis complex by strand displacement assay and a subset of specimens were examined for bacterial rRNA by fluorescent in situ hybridisation using an eubacterial probe with general bacterial specificity (EUB338). RESULTS: One patient with sarcoidosis displayed a positive M. tuberculosis complex test. All negative controls were negative in the test and 5/12 patients with mycobacteriosis were positive in the test. We detected M. tuberculosis complex DNA in 10-year-old biopsy samples. Thirty-six samples were tested with the eubacterial probe; of these, 67% were positive with no difference between patients and controls. CONCLUSION: Our results do not support the hypothesis that M. tuberculosis complex infection is involved in the pathogenesis of sarcoidosis. However, we stress the importance of excluding mycobacteriosis in the diagnostic workup of sarcoidosis patients.

Seasonal and habitat variation in the prevalence of Rickettsia helvetica in Ixodes ricinus ticks from Denmark.

A total of 704 unfed ticks of the species Ixodes ricinus collected in Denmark were screened for Rickettsia DNA by a genus-specific real-time PCR. Of the nymphs, 4.7% (31/662) were positive for rickettsial DNA. Among the positive ticks, we observed a seasonal and habitat variation. The infection rate was highest in May as compared to July, August, and October. Ecotone (high tick density) showed an elevated prevalence as compared to spruce or beech forests. Sequencing revealed only DNA from R. helvetica.

Health-related quality of life in adult survivors of childhood sarcoidosis.

AIM: To describe health-related quality of life (hrQOL) in adult subjects who had sarcoidosis in childhood. METHODS: Forty-six children (24 boys), all ethnic Danes

Detection of Rickettsia spp. in Danish ticks (Acari: Ixodes ricinus) using real-time PCR.

We examined 192 Ixodes ricinus tick nymphs from Denmark for the presence of Rickettsia spp. We used a species-specific real-time PCR; 13.0% of the ticks were positive for rickettsial DNA. Only DNA from Rickettsia helvetica was found in the ticks.