The Validation of a New Visual Anaemia Evaluation Tool HemoHue HH1 in Patients with End-Stage Renal Disease

In chronic haemodialysis patients, anaemia is a frequent finding associated with high therapeutic costs and further expenses resulting from serial laboratory measurements. HemoHue HH1, HemoHue Ltd, is a novel tool consisting of a visual scale for the noninvasive assessment of anaemia by matching the coloration of the conjunctiva with a calibrated hue scale. The aim of the study was to investigate the usefulness of HemoHue in estimating individual haemoglobin concentrations and binary treatment outcomes in haemodialysis patients. A prospective blinded study with 80 hemodialysis patients comparing the visual haemoglobin assessment with the standard laboratory measurement was performed. Each patient's haemoglobin concentration was estimated by seven different medical and nonmedical observers with variable degrees of clinical experience on two different occasions. The estimated population mean was close to the measured one (11.06 ± 1.67 versus 11.32 ± 1.23 g/dL, P < 0.0005). A learning effect could be detected. Relative errors in individual estimates reached, however, up to 50%. Insufficient performance in predicting binary outcomes (ROC AUC: 0.72 to 0.78) and poor interrater reliability (Kappa < 0.6) further characterised this method.

Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system

PURPOSE: To evaluate the expression and presence of surfactant protein (SP) A and SP-D in the lacrimal apparatus, at the ocular surface, and in tears in healthy and pathologic states. METHODS: Expression of mRNA for SP-A and SP-D was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts as well as in a spontaneously immortalized conjunctival epithelial cell line (HCjE; IOBA-NHC) and a SV40-transfected cornea epithelial cell line (HCE). Deposition of SP-A and SP-D was determined by Western blot, dot blot, and immunohistochemistry in healthy tissues, in tears, aqueous humor, and in sections of different corneal abnormalities (keratoconus, herpetic keratitis, and Staphylococcus aureus-based ulceration). Cell lines were stimulated with different cytokines and bacterial components and were analyzed for the production of SP-A and SP-D by immunohistochemistry. RESULTS: The presence of SP-A and SP-D on mRNA and protein levels was evidenced in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal duct samples. Moreover, both proteins were present in tears but were absent in aqueous humor. Immunohistochemistry revealed the production of both peptides by acinar epithelial cells of the lacrimal gland and epithelial cells of the conjunctiva and nasolacrimal ducts, whereas goblet cells revealed no reactivity. Healthy cornea revealed weak reactivity on epithelial surface cells only. In contrast, SP-A and SP-D revealed strong reactivity in patients with herpetic keratitis and corneal ulceration surrounding lesions and in several immigrated defense cells. Reactivity in corneal epithelium and endothelium was also seen in patients with keratoconus. Cell culture experiments revealed that SP-A and SP-D are produced by both epithelial cell lines without and after stimulation with cytokines and bacterial components. CONCLUSIONS: These results show that SP-A, in addition to SP-D, is a peptide of the tear film. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D seem to be involved in several ocular surface diseases.

Microcirculation in hypertensive patients

Regardless of the mechanisms that initiate the increase in blood pressure, functional and structural changes in the systemic vasculature are the final result of long-standing hypertension. These changes can occur in the macro- but also in the microvasculature. The supply of the tissues with oxygen, nutrients, and metabolites occurs almost exclusively in the microcirculation (which comprises resistance arterioles, capillaries and venules), and an adequate perfusion via the microcirculatory network is essential for the integrity of tissue and organ function. This review focuses on results from clinical studies in hypertensive patients, which have been performed in close cooperation with different clinical groups over the last three decades. Intravital microscopy was used to study skin microcirculation, microcatheters for the analysis of skeletal muscle microcirculation, the slit lamp for conjunctival microcirculation and the laser scanning ophthalmoscope for the measurement of the retinal capillary network. The first changes of the normal microcirculation can be found in about 93% of patients with essential hypertension, long before organ dysfunctions become clinically manifest. The earliest disorders were found in skin capillaries and thereafter in the retina and the skeletal muscle. In general, the disorders in the different areas were clearly correlated. While capillary rarefaction occurred mainly in the retina and the conjunctiva bulbi, in skin capillaries morphological changes were rare. A significant decrease of capillary erythrocyte velocities under resting conditions together with a marked damping of the postischemic hyperemia was found, both correlating with the duration of hypertension or WHO stage or the fundus hypertonicus stage. Also the mean oxygen tension in the skeletal muscle was correlated with the state of the disease. These data show that the microcirculatory disorders in hypertension are systemic and are hallmarks of the long-term complications of hypertension. There is now a large body of evidence that microvascular changes occur very early and may be important in their pathogenesis and progression.

Chronic bovine besnoitiosis: intra-organ parasite distribution, parasite loads and parasite-associated lesions in subclinical cases.

Bovine besnoitiosis caused by Besnoitia besnoiti is a chronic and debilitating disease. The most characteristic clinical signs of chronic besnoitiosis are visible tissue cysts in the scleral conjunctiva and the vagina, thickened skin and a generally poor body condition. However, many seropositive animals remain subclinically infected, and the role that these animals may play in spreading the disease is not known. The aim of the present study was to assess the intra-organ parasite distribution, the parasite load and the parasite-associated lesions in seropositive but subclinically infected animals. These animals were seropositive at the time of several consecutive samplings, had visible tissue cysts in the past and, at time of slaughter, had detectable specific anti-Besnoitia spp. antibody levels, but they did not show evident clinical signs at culling. Thus, histopathological, immunohistochemical and molecular analyses of several samples from the respiratory tract, reproductive tract, other internal organs and skin from six cows were performed. The tissue cysts were located primarily in the upper respiratory tract, i.e., in the rhinarium and larynx/pharynx (four cows), followed by the distal genital tract (vulva/vagina) and the skin of the neck (three and two cows, respectively, out of the four cows with cysts in the respiratory tract). We were unable to detect any parasites in the two remaining cows. Cysts were associated with a significant non-purulent inflammatory infiltrate consisting predominantly of T lymphocytes and activated monocytes/macrophages in two cows. The parasite burden, estimated by quantitative real-time PCR, was very low. It is noteworthy that the only animal that showed a recent increase in the antibody titre had the highest parasite burden and the most conspicuous inflammatory reaction against the cysts. In conclusion, although these cows no longer displayed any visible signs of besnoitiosis, they remained infected. Therefore, cows without visible signs of disease may still be able to transmit the parasite.

An inter-laboratory comparative study of serological tools employed in the diagnosis of Besnoitia besnoiti infection in bovines

Bovine besnoitiosis is considered an emerging chronic and debilitating disease in Europe. Many infections remain subclinical, and the only sign of disease is the presence of parasitic cysts in the sclera and conjunctiva. Serological tests are useful for detecting asymptomatic cattle/sub-clinical infections for control purposes, as there are no effective drugs or vaccines. For this purpose, diagnostic tools need to be further standardized. Thus, the aim of this study was to compare the serological tests available in Europe in a multi-centred study. A coded panel of 241 well-characterized sera from infected and non-infected bovines was provided by all participants (SALUVET-Madrid, FLI-Wusterhausen, ENV-Toulouse, IPB-Berne). The tests evaluated were as follows: an in-house ELISA, three commercial ELISAs (INGEZIM BES 12.BES.K1 INGENASA, PrioCHECK Besnoitia Ab V2.0, ID Screen Besnoitia indirect IDVET), two IFATs and seven Western blot tests (tachyzoite and bradyzoite extracts under reducing and non-reducing conditions). Two different definitions of a gold standard were used: (i) the result of the majority of tests ('Majority of tests') and (ii) the majority of test results plus pre-test information based on clinical signs ('Majority of tests plus pre-test info'). Relative to the gold standard 'Majority of tests', almost 100% sensitivity (Se) and specificity (Sp) were obtained with SALUVET-Madrid and FLI-Wusterhausen tachyzoite- and bradyzoite-based Western blot tests under non-reducing conditions. On the ELISAs, PrioCHECK Besnoitia Ab V2.0 showed 100% Se and 98.8% Sp, whereas ID Screen Besnoitia indirect IDVET showed 97.2% Se and 100% Sp. The in-house ELISA and INGEZIM BES 12.BES.K1 INGENASA showed 97.3% and 97.2% Se; and 94.6% and 93.0% Sp, respectively. IFAT FLI-Wusterhausen performed better than IFAT SALUVET-Madrid, with 100% Se and 95.4% Sp. Relative to the gold standard 'Majority of test plus pre-test info', Sp significantly decreased; this result was expected because of the existence of seronegative animals with clinical signs. All ELISAs performed very well and could be used in epidemiological studies; however, Western blot tests performed better and could be employed as a posteriori tests for control purposes in the case of uncertain results from valuable samples.

First cases of besnoitiosis in cattle in Switzerland

Bovine besnoitiosis has been diagnosed in neighboring countries but not in Switzerland so far. This disease occurs endemically in France and focal outbreaks have been reported in Germany and Italy. To determine if Besnoitia besnoiti is introduced into Switzerland through the import of breeding cattle from France, a systematic serological survey was performed. A total of 412 breeding cattle (from 114 farms) imported from France into Switzerland between 2005 and 2011, were serologically examined for antibodies against B. besnoiti using a commercial ELISA kit (PrioCHECK© Besnoitia Ab 2.0, Prionics AG, Zurich, Switzerland). Sixty-four (15.5 %) animals reacted positive in ELISA. The serologic diagnosis was confirmed by an indirect immunfluorescence test (IFAT) and a Western blot (WB) in only 2 Limousin cows imported from France on a farm in Eastern Switzerland. Subsequently, this whole herd (n = 16) was examined clinically and serologically and 2 additional Limousin cows imported from Germany also reacted positive in the three serological tests. One of these cows presented B. besnoiti tissue cysts in the scleral conjunctiva and typical skin lesions in the head region. The infection was further confirmed cytologically, histopathologically and by PCR. It can be concluded that the parasite is most likely being introduced into Switzerland through the import of infected animals.