Perspectives and challenges in validating new diagnostic technologies.

This paper focuses on several new diagnostic technologies, which are set to dominate the testing landscape in the near future and have applications in animal health diagnostics, namely: next-generation sequencing, assays to detect biomarkers, and point-of-care tests. An example of real-time loop-mediated isothermal amplification validation is also provided. Validating these new technologies presents several challenges, which are addressed in this paper.

Les auteurs s'intéressent à plusieurs nouvelles technologies de diagnostic appelées à occuper, dans un futur proche, une place de choix dans le paysage du dépistage et dont il existe déjà des applications en santé animale, à savoir : le séquençage de nouvelle génération, la détection de biomarqueurs et les tests utilisables sur le lieu des soins. Ils décrivent par ailleurs l'exemple de la validation d'une amplification isotherme à médiation par boucle en temps réel. La validation de ces nouvelles technologies présente un certain nombre de difficultés, que les auteurs examinent en détail.

Los autores se centran en varias tecnologías de nuevo cuño que están llamadas a dominar el panorama de las pruebas de diagnóstico en un futuro próximo y que tienen aplicaciones de diagnóstico en sanidad animal, a saber: la secuenciación de próxima generación, los ensayos de detección de marcadores biológicos y las pruebas practicadas en el lugar de consulta. También ofrecen un ejemplo de validación de una técnica de amplificación isotérmica mediada por bucles en tiempo real. La validación de estas nuevas tecnologías presenta varias dificultades, que los autores examinan en estas líneas.

Re-visiting the detection of porcine cysticercosis based on full carcass dissections of naturally Taenia solium infected pigs.

BACKGROUND: Taenia solium is a neglected zoonotic parasite. The performances of existing tools for the diagnosis of porcine cysticercosis need further assessment, and their shortcomings call for alternatives. The objective of this study was to evaluate the performance of tongue palpation and circulating antigen detection for the detection of porcine cysticercosis in naturally infected pigs of slaughter age compared to full carcass dissections (considered the gold standard). Additionally, alternative postmortem dissection procedures were investigated. A total of 68 rural pigs of slaughter age randomly selected in the Eastern Province of Zambia were dissected. Dissections were conducted on full carcasses (or half carcass in case cysticerci were already detected in the first half), including all the organs. Total cysticercus counts, location and stages were recorded and collected cysticerci were identified morphologically and molecularly. All sera were analysed with the B158/B60 antigen detecting ELISA (Ag-ELISA). RESULTS: Key findings were the high occurrence of T. solium infected pigs (56%) and the presence of T. solium cysticerci in the livers of 26% of infected animals. More than half of the infected carcasses contained viable cysticerci. Seven carcasses had T. hydatigena cysticerci (10%), out of which five carcasses were co-infected with T. hydatigena and T. solium; two carcasses (3%) had only T. hydatigena cysticerci. Compared to full carcass dissection, the specificity of the Ag-ELISA to detect infected carcasses was estimated at 67%, the sensitivity at 68%, increasing to 90% and 100% for the detection of carcasses with one or more viable cysticerci, and more than 10 viable cysts, respectively. Tongue palpation only detected 10% of the cases, half carcass dissection 84%. Selective dissection of the diaphragm, tongue and heart or masseters can be considered, with an estimated sensitivity of 71%, increasing to 86% in carcasses with more than 10 cysticerci. CONCLUSIONS: Depending on the aim of the diagnosis, a combination of Ag-ELISA and selective dissection, including investigating the presence of T. hydatigena, can be considered. Full carcass dissection should include the dissection of the liver, kidneys, spleen and lungs, and results should be interpreted carefully, as small cysticerci can easily be overlooked.

Placental iodothyronine deiodinase expression in normal and growth-restricted human pregnancies.

We have described the expression of specific iodothyronine deiodinase mRNAs (using quantitative RT-PCR) and activities in normal human placentas throughout gestation and compared our findings to those in placentas from pregnancies affected by intrauterine growth restriction (IUGR). The predominant deiodinase expressed in placenta was type III (D3); type II (D2) was also present. In general terms, the activities of the enzymes D2 and D3 (and mRNAs encoding these enzymes) were higher earlier in gestation (<28 wk) than at term and displayed an inverse relationship with the duration of gestation (P < 0.05). Comparison of the relative expressions of mRNAs encoding D2 and D3 as well as their activities in placentas associated with IUGR (early and late gestational groups) with findings from normal placentas of similar gestational ages revealed no significant differences. Immunolocalization of D2 and D3 in syncytiotrophoblast (including syncytial sprouts) and cytotrophoblast of human placentas was demonstrated at both early and late gestation. Treatment of primary cultures of term cytotrophoblast cells in vitro with increasing doses of T(3) (1, 10, and 100 nM) resulted in increased expression of mRNAs encoding both D2 and D3 at 100-nM concentrations (P < 0.01) compared with control. Experiments with JEG-3 choriocarcinoma cells demonstrated a similar effect on D3 mRNA at 10 and 100 nM T(3) (P < 0.01). The demonstrated changes in iodothyronine deiodinase expression in the placenta across pregnancy are likely to contribute to regulation of the thyroid hormone supply to the developing fetus. The lack of difference in deiodinase expression in normal placentas and those found in IUGR argues against placental deiodinases being responsible for the hypothyroxemia in circulating fetal thyroid hormones observed in this condition.

Costal fringe.

Costal fringe consists of a bandlike pattern of venous telangiectases along the anterolateral costal margins. It is a common and easily recognized entity, usually seen in elderly men. Association with underlying disease is not well established. To assess the frequency of costal fringe in a healthy young adult population, we examined 1523 active duty Air Force personnel (1203 men and 320 women) between the ages of 17 and 34 years. All subjects were human immunodeficiency virus seronegative; none had known hyperestrogenic or other disease states. Costal fringe was present in only three individuals, all men, a frequency of 0.2%. Costal fringe is a rare finding in healthy young adults.

Efficacy of minocycline compared with tetracycline in treatment of acne vulgaris.

A double-blind evaluation of the efficacy and safety of minocycline hydrochloride and tetracycline hydrochloride was conducted and completed using 49 patients with Pillsbury grade 2 or grade 3 acne. For six months, half of the patients received minocycline and half received tetracycline. Although the differences between treatment groups were not statistically significant at any evaluation, more patients treated with minocycline reached and maintained a noninflammatory acne status in less time than did patients treated with tetracycline. After six weeks, twice as many patients in the group treated with minocycline had reached noninflammatory status. Side effects reported by 7 patients were equally distributed between treatment groups. No notable abnormalities were observed in the results of blood chemistry studies, hematologic tests, quantitative serum immunoglobulin determinations, or thyroid function tests in 20 of the patients examined.

Teratological evaluation of gamma-glycidoxypropyltrimethoxysilane in rats.

The embryotoxic and teratogenic potential of gamma- glycidoxypropyltrimethoxysilane ( GPTS ) was evaluated in rats. Pregnant Sprague-Dawley rats were administered 0, 50, 500, or 1,000 mg/kg/day of GPTS by gavage on days 6 through 15 of gestation. No treatment related signs of toxicity, behavioral alterations or mortalities were observed in any of the pregnant animals. There was no evidence of adverse effects in mean maternal body weight, liver weight or food consumption of the treated females. The number of implantation sites, number of live fetuses per litter, the mean litter size, the sex ratio, the fetal body weight or the crown-rump length were not affected by treatment. The incidence of resorptions among the total fetal population was not altered by the administration of GPTS to pregnant rats, indicating that the test material is not embryolethal in rats at the tested dose levels. Few scattered incidences of fetal alterations in the external, soft tissue or skeletal examinations were seen both among treated and untreated litters, however, no single alteration was observed in treated litters at an incidence which was significantly different from the control. In conclusion GPTS was not embryotoxic or teratogenic in rats at dose levels up to 1000 mg/kg/day.

Interstitial pulmonary edema in children and adolescents with diabetic ketoacidosis.

The acute complications of diabetic ketoacidosis in children and adolescents are well recognized but not completely understood. Clinical studies have focused primarily on brain edema. We have investigated the prevalence and course of interstitial pulmonary edema in patients with severe diabetic ketoacidosis all of whom had uneventful clinical courses. High resolution computed tomography scans of the lungs were analyzed by determining the Hounsfield attenuation level and then converting to physical density values. All seven patients had evidence of interstitial pulmonary edema on the first scan, which was performed within 1 h of hydration and prior to receiving insulin; six of the seven patients had increased pulmonary density 6-8 h into treatment, and all had complete resolution of the interstitial changes at discharge. Our study suggests that subclinical interstitial pulmonary edema may be a frequent occurrence in children and adolescents with severe diabetic ketoacidosis and may very well be present prior to treatment. The study also supports the philosophy of cautious rehydration and the close monitoring of children and adolescents with diabetic ketoacidosis until a more complete understanding of this pathophysiologic event is achieved.

Coccidioidomycosis.

Although rapid population growth in the Southwestern United States and travel to and through the area are increasing the potential for exposure to Coccidioides immitis, prevalence rates have declined in some endemic areas, probably because of environmental factors. With the iatrogenic immunosuppression of organ transplantation and the immunosuppression inherent in AIDS, more opportunistic infections with this organism are to be expected. The variety of cutaneous manifestations continues to challenge the dermatologist's acumen. Spherule-derived coccidioidin is an improved epidemiologic tool, and serodiagnostic techniques are easier to perform and are useful in the management of dissemination. While amphotericin B remains the standard, ketoconazole has found a definite role in the treatment of this disease in many patients. Itraconazole, now under investigation, appears very promising. Morbidity and mortality from disseminated disease appear to be declining. With current diagnostic and therapeutic methods, the prognosis for survival in immunocompetent patients is excellent.

Electrosurgical epilation.

This article discusses alternative methods for the removal of unwanted hair, the mechanisms of action of each, and their advantages and disadvantages. Epilating needle design is discussed and epilating technique is described. Physician-patient interaction and the role of the dermatologist in electrosurgical epilation are explored.

The human placenta expresses multiple glucocorticoid receptor isoforms that are altered by fetal sex, growth restriction and maternal asthma.

INTRODUCTION: We have previously identified sex-specific differences in the fetal-placental response to cortisol. Our recent studies suggest that this differential response to cortisol is driven by differences in glucocorticoid receptor (GR) protein function rather than through changes in gene transcription or protein expression. METHODS: This study was designed to define whether the human placenta expresses different isoforms of the GR and whether expression was altered by fetal sex and maternal asthma. Asthma and non-asthma pregnant women were prospectively recruited at their first antenatal visit and placentae collected at delivery. Placental GR expression was examined in relation to maternal asthma, fetal sex and birthweight. RESULTS: Twelve specific bands for the GR were identified at molecular weights of 94, 91, 81, 74, 69, 68, 65, 60, 55, 50, 48 and 38 kDa. The 12 isoforms were localised to the placental trophoblast and expression varied in relation to cellular location in either the cytoplasm or nucleus, fetal sex, fetal size and the presence and absence of maternal asthma. CONCLUSION: This is the first study to identify the presence of several protein isoforms of the GR in the human placenta. The data suggest glucocorticoid resistance observed in male placentae may be mediated through increased GRß, GR A and GR P localisation to the nucleus. While female placentae may be more sensitive to cortisol in the presence of maternal asthma through a decrease in GRß and an enhancement GRα activity via an interaction with GRα D3 and GRα C.

Akt1 deletion prevents lung tumorigenesis by mutant K-ras.

K-ras mutations are associated with smoking-induced lung cancer and poor clinical outcomes. In mice, K-ras mutations are sufficient to induce lung tumors, which require phosphoinoside-3-kinase (PI3K) and further downstream, mammalian target of rapamycin (mTOR) activation. However, the roles of individual Akt isoforms that link PI3K and mTOR are unknown. Here, we show that deletion of Akt1 but not Akt2 or Akt3 prevents lung tumorigenesis in a tobacco carcinogen-induced model and a genetic model. Akt1 deletion prevented tumor initiation as well as tumor progression, coincident with decreased Akt signaling in tumor tissues. In contrast, deletion of Akt3 increased tumor multiplicity in the carcinogen model and increased tumor size in the genetic model. Fibroblasts lacking Akt1 are resistant to transformation by mutant K-ras and stimulation by epidermal growth factor. Human lung cancer cells with mutant K-ras and diminished Akt1 levels fail to grow in vivo. These data suggest that Akt1 is the primary Akt isoform activated by mutant K-ras in lung tumors, and that Akt3 may oppose Akt1 in lung tumorigenesis and lung tumor progression. Given that Akt inhibitors in clinical development as cancer therapeutics are not isoform selective, these studies support specific targeting of Akt1 to mitigate the effects of mutant K-ras in lung cancer.

Argon laser treatment of angiokeratomas.

The argon laser, used in either a pulsed or continuous fashion, is highly effective for treatment of angiokeratomas. Local anesthesia is usually not required. Because energy deposition is well localized, wounds heal rapidly with excellent cosmetic results.

Treatment of multiple dermal cylindromas with the carbon dioxide laser.

The management of multiple cylindromas presents a therapeutic challenge to the clinician. The condition has significant cosmetic implications, and malignant degeneration can occur. Reported treatments include excision and repair of individual lesions, high-dose radiation, and complete scalp removal with grafting. We review the proposed histogenesis of this tumor, discuss its associations, and report the first use of the carbon dioxide laser as a successful treatment.

Subchronic oral toxicity study of gamma-glycidoxypropyltrimethoxysilane in rats.

The subchronic toxicity (28 days) of orally administered gamma-Glycidoxypropyltrimethoxysilane (GPTS) was studied in laboratory rats. The test material was administered daily for four weeks to groups of 10 male and 10 female Sprague-Dawley rats by gavage at dose levels of 0, 40, 400, and 1,000 mg/kg. Mortality, behavioral reactions, growth and food consumption were observed and measured along with hematology, blood biochemistry, absolute and relative organ weights. No overt signs of toxicity or behavioral abnormalities were observed in any of the test animals during the course of the study. There were no treatment related mortalities and no significant differences were observed in mean body weight, food consumption, absolute or relative organ weights of control and treated rats. Also, there were no meaningful differences in hematology, urinalysis or clinical blood chemistry values between control and treated animals. Gross and histopathologic examinations of organs or tissues from both control and GPTS treated animals did not reveal any treatment related changes. These results suggest that it is unlikely that serious injury would result from the ingestion of GPTS in amounts normally encountered incidental to its industrial use.

PRISM: protein integration of sequence metrics.

PRISM is a comprehensive method for predicting protein sequence structure and function by providing a mechanism to combine information from numerous structure and function prediction tools into one comprehensive view. It uses an evidential reasoning calculus to combine information provided from multiple sources into a consensus solution. The output of PRISM is a comprehensive one stop shop report of all information about a given known or novel protein.

Subchronic dermal toxicity study of allyl methacrylate in rabbits.

A 28-day subchronic dermal toxicity of allyl methacrylate (AMA) was conducted in New Zealand white rabbits. Groups of six male and six female rabbits were treated with the test material at dose levels of 0, 25, 50 and 100 mg/kg/day for four weeks. One satellite group of male and female rabbits was also treated with 100 mg/kg/day of AMA. No overt signs of toxicity or abnormal behavior were seen among the rabbits during the treatment period. Two females from the 50 mg/kg/day group and two females at the highest dose level died during the course of the study. No mortalities and no significant adverse effects were observed in the low dose and control rabbits. Males treated with the highest dose level exhibited reduced weight gain and lower food consumption. The test material has no significant adverse effects on serum biochemistry, urine and hematological parameters or absolute and relative organ weights. No chemical related gross pathological alterations were observed in any of the organs or tissues examined at the time of necropsy except slight hemorrhage in the fascia of the skin of rabbits treated with 100 mg/kg/day of AMA. There were no significant chemical related microscopic changes found in any of the test rabbits. The animals of the satellite group appeared fully recovered following the 28-day dermal exposure to AMA. These results suggest that it is unlikely that AMA will present a significant health hazard from skin contact under normal conditions of industrial handling.

Subchronic dermal toxicity of trifluoropropylmethylcyclotrisiloxane in rabbits.

A 21-day subchronic dermal toxicity of trifluoropropylmethylcyclotrisiloxane (TFP) was conducted in New Zealand white rabbits. One control and three treatment groups of male and female animals were administered with the test material at levels of 0, 40, 200 and 400 mg/kg/day for three weeks. Mortality, behavioral reactions, growth and food consumption were observed and measured along with hematology, clinical blood chemistry, relative organ weight and pathology parameters. Five animals died at the highest dose level during the test period. Treatment with two higher dosages generally resulted in reduced rate of weight gain, lower food consumption, depressed serum alkaline phosphatase activity and increased activity of the serum enzymes. No significant adverse effects were observed at lower levels of 40 mg/kg. Gross pathologic examination revealed no treatment related causes of death and all organs and tissues were observed normal at the time of necropsy. There were no chemical related microscopic changes found in any of the test animals.

Enhancement of the clinical margins of Bowen's disease by acetowhitening.

Acetic acid was applied preoperatively to 12 lesions of Bowen's disease to determine if acetowhitening could more clearly define clinical margins. Acetowhitening disclosed the true margins better than unenhanced direct visualization, and subclinical extension of Bowen's disease was revealed by acetowhitening in three lesions. Acetowhitening can be a useful adjunct in the surgical management of Bowen's disease.