Species-specific and Indication-based Use of Antimicrobials in Dogs, Cats, Cattle and Horses in Finland : Data collected using three different methods

Increasing antimicrobial resistance in bacteria has led to the need for better understanding of antimicrobial usage patterns. In 1999, the World Organisation for Animal Health (OIE) recommended that an international ad hoc group should be established to address human and animal health risks related to antimicrobial resistance and the contribution of antimicrobial usage in veterinary medicine. In European countries the need for continuous recording of the usage of veterinary antimicrobials as well as for animal species-specific and indication-based data on usage has been acknowledged. Finland has been among the first countries to develop prudent use guidelines in veterinary medicine, as the Ministry of Agriculture and Forestry issued the first animal species-specific indication-based recommendations for antimicrobial use in animals in 1996. These guidelines have been revised in 2003 and 2009. However, surveillance on the species-specific use of antimicrobials in animals has not been performed in Finland. This thesis provides animal species-specific information on indication-based antimicrobial usage. Different methods for data collection have been utilized. Information on antimicrobial usage in animals has been gathered in four studies (studies A-D). Material from studies A, B and C have been used in an overlapping manner in the original publications I-IV. Study A (original publications I & IV) presents a retrospective cross-sectional survey on prescriptions for small animals at the Veterinary Teaching Hospital of the University of Helsinki. Prescriptions for antimicrobial agents (n = 2281) were collected and usage patterns, such as the indication and length of treatment, were reviewed. Most of the prescriptions were for dogs (78%), and primarily for the treatment of skin and ear infections most of which were treated with cephalexin for a median period of 14 days. Prescriptions for cats (18%) were most often for the treatment of urinary tract infections with amoxicillin for a median length of 10 days. Study B (original publication II) was a retrospective cross-sectional survey where prescriptions for animals were collected from 17 University Pharmacies nationwide. Antimicrobial prescriptions (n = 1038) for mainly dogs (65%) and cats (19%) were investigated. In this study, cephalexin and amoxicillin were also the most frequently used drugs for dogs and cats, respectively. In study C (original publications III & IV), the indication-based usage of antimicrobials of practicing veterinarians was analyzed by using a prospective questionnaire. Randomly selected practicing veterinarians in Finland (n = 262) recorded all their antimicrobial usage during a 7-day study period. Cattle (46%) with mastitis were the most common patients receiving antimicrobial treatment, generally intramuscular penicillin G or intramammary treatment with ampicillin and cloxacillin. The median length of treatment was four days, regardless of the route of administration. Antimicrobial use in horses was evaluated in study D, the results of which are previously unpublished. Firstly, data collected with the prospective questionnaire from the practicing veterinarians showed that horses (n = 89) were frequently treated for skin or wound infections by using penicillin G or trimethoprim-sulfadiazine. The mean duration of treatment was five to seven days. Secondly, according to retrospective data collected from patient records, horses (n = 74) that underwent colic surgery at the Veterinary Teaching Hospital of the University of Helsinki were generally treated according to national and hospital recommendations; penicillin G and gentamicin was administered preoperatively and treatment was continued for a median of three days postoperatively. In conclusion, Finnish veterinarians followed well the national prudent use guidelines. Narrow-spectrum antimicrobials were preferred and, for instance, fluoroquinolones were used sparingly. Prescription studies seemed to give good information on antimicrobials usage, especially when combined with complementary information from patient records. A prospective questionnaire study provided a fair amount of valuable data on several animal species. Electronic surveys are worthwhile exploiting in the future.

Detection,amplification and sequence homology of sodC in clinical isolates of Salmonella sp

Background & objectives: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified, cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were compared with the standard strain Salmonella Typhimurium 14028. Methods: Salmonella enterica serovar Typhi (S. Typhi) and Salmonellaenterica serovar Paratyphi (S. Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC) was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were compared using CLUSTAL X.Results: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons respectively. Single mutational difference at position 489 was observed between thesodC of S. Typhi and S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that of standard strain S. Typhimurium 14028.Interpretation & conclusions: The presence of SodC in pathogenic bacteria could be a novel candidate as phylogenetic marker.

Consequences of Vertebral Fractures

Vertebral fractures occur due to forces applied to spinal structures. When the bone tissue is weakened, vertebral fractures can result from a minor trauma. Adult vertebral fractures are commonly considered to be an indication for osteoporosis. In children osteoporosis is a rare condition, and pediatric vertebral fractures are usually clearly trauma-related. The aims of this dissertation are to produce knowledge of the epidemiology of osteoporotic vertebral fractures and to analyse their association with total and cause-specific mortality, to find indicators with which to identify individuals who are at great risk of subsequent fractures, to study the incidence of pediatric vertebral fractures and need for their operative treatment and hospital care. The Mobile-Clinic and Mini-Finland Health surveys of the adult population were used as materials in this research. Record linkages to the Finnish Hospital Discharge Register and the Official Cause of Death register were used to study mortality and hospitalization in the same population group. These registers were also used to evaluate epidemiology, mortality, hospitalization and the need for operative management of pediatric vertebral fracture patients. The main findings and conclusions of the present dissertation are: 1. The presence of a thoracic vertebral fracture in adults is a significant predictor of cancer and respiratory mortality. In women, but not in men, vertebral fractures strongly predict mortality due to injuries. Most of these deaths in the study group were hip fracture related. 2. Severe thoracic vertebral fracture in adults was a strong predictor of a subsequent hip fracture, whereas mild or moderate fractures and the number of compressed vertebrae were much weaker predictors, 3. Pediatric spinal fractures were rare: The incidence was 66 per one million children per year. In younger children cervical spine was most often affected, whereas in older children fractures of the thoracic and lumbar spine were more common. Maturation of spinal structures seems to play a major role in the typical injury patterns in children. Thirty per cent of pediatric spinal fractures required surgical treatment. The current study focuses on consequences of vertebral fractures in general, without evaluating further the causation of the studied phenomena. Further studies are needed to clarify the mechanisms of association between vertebral fractures and specific causes of mortality. A severe vertebral fracture appears to indicate a substantial risk of a subsequent hip fracture. If such a fracture is identified from a chest radiograph, urgent clinical evaluation, treatment of osteoporosis and protective measures against falls are recommended.

Molecular and Clinical Characteristics of Pituitary Adenoma Predisposition (PAP)

Pituitary adenomas are common benign neoplasms. Although most of them are sporadic, a minority occurs in familial settings. Heterozygous germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were found to underlie familial pituitary adenomas, a condition designated as pituitary adenoma predisposition (PAP). PAP confers incomplete penetrance of mostly growth hormone (GH) secreting adenomas in young patients, who often lack a family history of pituitary adenomas. This thesis work aimed to clarify the molecular and clinical characteristics of PAP. Applying the multiplex ligation-dependent probe amplification assay (MLPA), we found large genomic AIP deletions to account for a subset of PAP. Therefore, MLPA could be considered in PAP suspected patients with no AIP mutations found with conventional sequencing. We generated an Aip mouse model to examine pituitary tumorigenesis in vivo. The heterozygous Aip mutation conferred complete penetrance of pituitary adenomas that were mostly GH-secreting, rendering the phenotype of the Aip mouse similar to that of PAP patients. We suggest that AIP may function as a candidate gatekeeper gene in somatotrophs. To clarify molecular mechanisms of tumorigenesis, we elucidated the expression of AIP-related molecules in human and mouse pituitary tumors. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT) was reduced in mouse Aip-deficient adenomas, and similar ARNT reduction was also evident in human AIP mutation positive adenomas. This suggests that in addition to participating in the hypoxia pathway, estrogen receptor signaling and xenobiotic response pathways, ARNT may play a role in AIP-related tumorigenesis. We also studied the characteristics and the response to therapy of PAP patients and found them to have an aggressive disease phenotype with young age at onset. Therefore, improvement in treatment outcomes of PAP patients would require their efficient identification and earlier diagnosis of the pituitary adenomas. The possible role of the RET proto-oncogene in tumorigenesis of familial AIP mutation negative pituitary adenomas was evaluated, but none of the found RET germline variants were considered pathogenic. Surprisingly, RET immunohistochemistry suggested possible underexpression of RET in AIP mutation positive pituitary adenomas an observation that merits further investigation.

Excimer laser refractive surgery : corneal wound healing and clinical results

Although the first procedure in a seeing human eye using excimer laser was reported in 1988 (McDonald et al. 1989, O'Connor et al. 2006) just three studies (Kymionis et al. 2007, O'Connor et al. 2006, Rajan et al. 2004) with a follow-up over ten years had been published when this thesis was started. The present thesis aims to investigate 1) the long-term outcomes of excimer laser refractive surgery performed for myopia and/or astigmatism by photorefractive keratectomy (PRK) and laser-in situ- keratomileusis (LASIK), 2) the possible differences in postoperative outcomes and complications when moderate-to-high astigmatism is treated with PRK or LASIK, 3) the presence of irregular astigmatism that depend exclusively on the corneal epithelium, and 4) the role of corneal nerve recovery in corneal wound healing in PRK enhancement. Our results revealed that in long-term the number of eyes that achieved uncorrected visual acuity (UCVA)≤0.0 and ≤0.5 (logMAR) was higher after PRK than after LASIK. Postoperative stability was slightly better after PRK than after LASIK. In LASIK treated eyes the incidence of myopic regression was more pronounced when the intended correction was over >6.0 D and in patients aged <30 years.Yet the intended corrections in our study were higher for LASIK than for PRK eyes. No differences were found in percentages of eyes with best corrected visual acuity (BCVA) or loss of two or more lines of visual acuity between PRK and LASIK in the long-term. The postoperative long-term outcomes of PRK with two different delivery systems broad beam and scanning laser were compared and revealed no differences. Postoperative outcomes of moderate-to-high astigmatism yielded better results in terms of UCVA and less compromise or loss of two more lines of BCVA after LASIK that after PRK.Similar stability for both procedures was revealed. Vector analysis showed that LASIK outcomes tended to be more accurate than PRK outcomes, yet no statistically differences were found. Irregular astigmatism secondary to recurrent corneal erosion due to map-dot-fingerprint was successfully treated with phototherapeutic keratectomy (PTK). Preoperative videokeratographies (VK) showed irregular astigmatism. However, postoperatively, all eyes showed a regular pattern. No correlation was found between pre- and postoperative VK patterns. Postoperative outcomes of late PRK in eyes originally subjected to LASIK showed that all (7/7) eyes achieved UCVA ≤0.5 at last follow-up (range 3 — 11 months), and no eye lost lines of BCVA. Postoperatively all eyes developed and initial mild haze (0.5 — 1) into the first month. Yet, at last follow-up 5/7 eyes showed a haze of 0.5 and this was no longer evident in 2/7 eyes. Based on these results, we demonstrated that the long-term outcomes after PRK and LASIK were safe and efficient, with similar stability for both procedures. The PRK outcomes were similar when treated by broad-beam or scanning slit laser. LASIK was better than PRK to correct moderate-to-high astigmatism, yet both procedures showed a tendency of undercorrection. Irregular astigmatism was proven to be able to depend exclusively from the corneal epithelium. If this kind of astigmatism is present in the cornea and a customized PRK/LASIK correction is done based on wavefront measurements an irregular astigmatism may be produced rather than treated. Corneal sensory nerve recovery should have an important role in the modulation of the corneal wound healing and post-operative anterior stromal scarring. PRK enhancement may be an option in eyes with previous LASIK after a sufficient time interval that in at least 2 years.

Detection of clinical mastitis with the help of a thermal camera

Increasing dairy farm size and increase in automation in livestock production require that new methods are used to monitor animal health. In this study, a thermal camera was tested for its capacity to detect clinical mastitis. Mastitis was experimentally induced in 6 cows with 10 mu g of Escherichia coli lipopolysaccharide (LPS). The LPS was infused into the left forequarter of each cow, and the right forequarters served as controls. Clinical examination for systemic and local signs and sampling for indicators of inflammation in milk were carried out before morning and evening milking throughout the 5-d experimental period and more frequently on the challenge day. Thermal images of experimental and control quarters were taken at each sampling time from lateral and medial angles. The first signs of clinical mastitis were noted in all cows 2 h postchallenge and included changes in general appearance of the cows and local clinical signs in the affected udder quarter. Rectal temperature, milk somatic cell count, and electrical conductivity were increased 4 h postchallenge and milk N-acetyl-beta-D-glucosaminidase activity 8 h postchallenge. The thermal camera was successful in detecting the 1 to 1.5 degrees C temperature change on udder skin associated with clinical mastitis in all cows because temperature of the udder skin of the experimental and control quarters increased in line with the rectal temperature. Yet, local signs on the udder were seen before the rise in udder skin and body temperature. The udder represents a sensitive site for detection of any febrile disease using a noninvasive method. A thermal camera mounted in a milking or feeding parlor could detect temperature changes associated with clinical mastitis or other diseases in a dairy herd.

Immediate hypersensitivity to Parthenium hysterophorus. II. Clinical studies on the prevalence of parthenium rhinitis

The airborne pollen of the South American weed, Parthenium hysterophorus (American feverfew), accidentally introduced into India was found to be responsible for severe allergic rhinitis. A random clinical survey conducted on 2035 residents of Bangalore city with the aid of questionnaires and skin tests revealed that 7.1% of the study population was suffering from allergic rhinitis due to exposure to Parthenium pollen. Skin-prick tests performed on 1294 clinic patients suffering from nasobronchial allergy during the past 4 years have also shown that 42.5% were sensitive to Parthenium pollen. IgE and IgG antibodies specific for Parthenium pollen allergens were demonstrable in the sera of Parthenium-sensitive rhinitis patients. The specificity of these antibodies to Parthenium allergens was established by ELISA. A 7- to 11-fold higher stimulation was observed when lymphocytes from two Parthenium-sensitive rhinitis patients were treated in vitro with Parthenium pollen extract. To our knowledge, nowhere in the world has such a high incidence of allergic rhinitis due to a single pollen ever been reported.

Pain measurement and management in elderly patients : Clinical studies in long term hospital care and after cardiac surgery

The proportion of patients over 75 years of age, receiving all different types of healthcare, is constantly increasing. The elderly undergo surgery and anaesthetic procedures more often than middle-aged patients. Poor pain management in the elderly is still an issue. Although the elderly consumes the greatest proportion of prescribed medicines in Western Europe, most clinical pharmacological studies have been performed in healthy volunteers or middle-aged patients. The aim of this study was to investigate pain measurement and management in cognitively impaired patients in long term hospital care and in cognitively normal elderly patients after cardiac surgery. This thesis incorporated 366 patients, including 86 home-dwelling or hospitalized elderly with chronic pain and 280 patients undergoing cardiac surgery with acute pain. The mean age of patients was 77 (SD ± 8) years and approximately 8400 pain measurements were performed with four pain scales: Verbal Rating Scale (VRS), the Visual Analogue Scale (VAS), the Red Wedge Scale (RWS), and the Facial Pain Scale (FPS). Cognitive function, depression, functional ability in daily life, postoperative sedation and postoperative confusion were assessed with MMSE, GDS, Barthel Index, RASS, and CAM-ICU, respectively. The effects and plasma concentrations of fentanyl and oxycodone were measured in elderly (≥ 75 years) and middle-aged patients (≤ 60 years) and the opioid-sparing effect of pregabalin was studied after cardiac surgery. The VRS pain scores after movement correlated with the Barthel Index. The VRS was most successful in the groups of demented patients (MMSE 17-23, 11-16 and ≤ 10) and in elderly patients on the first day after cardiac surgery. The elderly had a higher plasma concentration of fentanyl at the end of surgery than younger patients. The plasma concentrations of oxycodone were comparable between the groups. Pain intensity on the VRS was lower and the sedation scores were higher in the elderly. Total oxycodone consumption during five postoperative days was reduced by 48% and the CAM-ICU scores were higher on the first postoperative day in the pregabalin group. The incidence of postoperative pain during movement was lower in the pregabalin group three months after surgery. This investigation demonstrates that chronic pain did not seem to impair daily activities in home-dwelling Finnish elderly. The VRS appeared to be applicable for elderly patients with clear cognitive dysfunction (MMSE ≤17) and it was the most feasible pain scale for the early postoperative period after cardiac surgery. After cardiac surgery, plasma concentrations of fentanyl in elderly were elevated, although oxycodone concentrations were at similar level compared to middle-aged patients. The elderly had less pain and were more sedated after doses of oxycodone. Therefore, particular attention must be given to individual dosing of the opioids in elderly surgical patients, who often need a smaller amount for adequate analgesia than middle-aged patients. The administration of pregabalin reduced postoperative oxycodone consumption after cardiac surgery. Pregabalin-treated patients had less confusion, and additionally to less postoperative pain on the first postoperative day and during movement at three months post-surgery. Pregabalin might be a new alternative as analgesic for acute postoperative and chronic pain management in the elderly. Its clinical role and safety remains to be verified in large-scale randomized and controlled studies. In the future, many clinical trials in the older category of patients will be needed to facilitate improvements in health care methods.

Growth mechanism of phases, Kirkendall voids, marker plane position, and indication of the relative mobilities of the species in the interdiffusion zone

Often, wrong conclusions about the mobilities of species are drawn from the position of the Kirkendall marker plane or voids in the interdiffusion zone. To clarify, I have discussed the growth mechanism of the phases and the position of the marker plane depending on the relative mobilities of the species. The formation of different kinds of voids in the interdiffusion zone is discussed. Further, the microstructure that could be found because of the Kirkendall effect is also explained.

Clinical Proteomics of the Neglected Human Malarial Parasite Plasmodium vivax

Recent reports highlight the severity and the morbidity of disease caused by the long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties in the laboratory-propagation of P. vivax, the biology of this parasite has not been adequately explored. While the proteome of P. falciparum, the causative agent of cerebral malaria, has been extensively explored from several sources, there is limited information on the proteome of P. vivax. We have, for the first time, examined the proteome of P. vivax isolated directly from patients without adaptation to laboratory conditions. We have identified 153 proteins from clinical P. vivax, majority of which do not show homology to any previously known gene products. We also report 29 new proteins that were found to be expressed in P. vivax for the first time. In addition, several proteins previously implicated as anti-malarial targets, were also found in our analysis. Most importantly, we found several unique proteins expressed by P. vivax. This study is an important step in providing insight into physiology of the parasite under clinical settings.

Immunomodulation using agonists and antagonists: potential clinical applications

Introduction: Extensive studies have gone into understanding the differential role of the innate and adaptive arms of the immune system in the context of various diseases. Receptor-ligand interactions are responsible for mediating cross-talk between the innate and adaptive arms of the immune system, so as to effectively counter the pathogenic challenge. While TLRs remain the best studied innate immune receptor, many other receptor families are now coming to the fore for their role in various pathologies. Research has focused on the discovery of novel agonists and antagonists for these receptors as potential therapeutics. Areas covered: In this review, we present an overview of the recent advances in the discovery of drugs targeting important receptors such as G-protein coupled receptors, TRAIL-R, IL-1 beta receptor, PPARs, etc. All these receptors play a critical role in the modulation of the immune response. We focus on the recent paradigms applied for the generation of specific and effective therapeutics for these receptors and their status in clinical trials. Expert opinion: Non-specific activation by antagonist/agonist is a difficult problem to dodge. This demands innovation in ligand designing with the use of strategies such as allosterism and dual-specific ligands. Rigorous preclinical and clinical studies are required in transforming a compound to a therapeutic.

IDH1 Mutations in Diffusely Infiltrating Astrocytomas Grade Specificity, Association With Protein Expression, and Clinical Relevance

IDH1 mutations are frequent genetic alterations in low-grade diffuse gliomas and secondary glioblastoma (GBM). To validate mutation frequency, IDH1 gene at codon 132 was sequenced in 74 diffusely infiltrating astrocytomas: diffuse astrocytoma (DA; World Health Organization WHO] grade II), anaplastic astrocytoma (AA; WHO grade III), and GBM (WHO grade IV). All cases were immunostained with IDH1-R132H monoclonal antibody. Mutational status was correlated with mutant protein expression, patient age, duration of symptoms, and prognosis of patients with GBM. We detected 31 (41.9%) heterozygous IDH1 mutations resulting in arginine-to-histidine substitution (R132H;CGT-CAT). All 12 DAs (100%), 13 of 14 AAs (92.9%), and 6 of 48 GBMs (12.5%) (5/6 83.3%] secondary, and 1/42 2.4%] primary) harbored IDH1 mutations. The correlation between mutational status and protein expression was significant (P < .001). IDH1 mutation status, though not associated with prognosis of patients with GBM, showed significant association with younger age and longer duration of symptoms in the whole cohort (P < .001). Our study validates IDH1 mutant protein expression across various grades of astrocytoma, and demonstrates a high incidence of IDH1 mutations in DA, AA, and secondary GBM.

Rationalization and prediction of drug resistant mutations in targets for clinical anti-tubercular drugs

Resistance to therapy limits the effectiveness of drug treatment in many diseases. Drug resistance can be considered as a successful outcome of the bacterial struggle to survive in the hostile environment of a drug-exposed cell. An important mechanism by which bacteria acquire drug resistance is through mutations in the drug target. Drug resistant strains (multi-drug resistant and extensively drug resistant) of Mycobacterium tuberculosis are being identified at alarming rates, increasing the global burden of tuberculosis. An understanding of the nature of mutations in different drug targets and how they achieve resistance is therefore important. An objective of this study is to first decipher sequence as well as structural bases for the observed resistance in known drug resistant mutants and then to predict positions in each target that are more prone to acquiring drug resistant mutations. A curated database containing hundreds of mutations in the 38 drug targets of nine major clinical drugs, associated with resistance is studied here. Mutations have been classified into those that occur in the binding site itself, those that occur in residues interacting with the binding site and those that occur in outer zones. Structural models of the wild type and mutant forms of the target proteins have been analysed to seek explanations for reduction in drug binding. Stability analysis of an entire array of 19 mutations at each of the residues for each target has been computed using structural models. Conservation indices of individual residues, binding sites and whole proteins are computed based on sequence conservation analysis of the target proteins. The analyses lead to insights about which positions in the polypeptide chain have a higher propensity to acquire drug resistant mutations. Thus critical insights can be obtained about the effect of mutations on drug binding, in terms of which amino acid positions and therefore which interactions should not be heavily relied upon, which in turn can be translated into guidelines for modifying the existing drugs as well as for designing new drugs. The methodology can serve as a general framework to study drug resistant mutants in other micro-organisms as well.

Clinical phenotype and the lack of mutations in the CHRNG, CHRND, and CHRNA1 genes in two Indian families with Escobar syndrome

The objective of this study was to report the clinical phenotype and genetic analysis of two Indian families with Escobar syndrome (ES). The diagnosis of ES in both families was made on the basis of published clinical features. Blood samples were collected from members of both families and used in genomic DNA isolation. The entire coding regions and intron-exon junctions of the ES gene CHRNG (cholinergic receptor, nicotinic, gamma), and two other related genes, CHRND and CHRNA1, were amplified and sequenced to search for mutations in both families. Both families show a typical form of ES. Sequencing of the entire coding regions including the intron-exon junctions of the three genes did not yield any mutations in these families. In conclusion, it is possible that the mutations in these genes are located in the promoter or deep intronic regions that we failed to identify or the ES in these families is caused by mutations in a different gene. The lack of mutations in CHRNG has also been reported in several families, suggesting the possibility of at least one more gene for this syndrome. Clin Dysmorphol 22:54-58 (C) 2013 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.