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.

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.

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.

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.

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.

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.

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.

Subchronic inhalation toxicity of tetramethoxysilane in rats.

Sprague-Dawley rats were exposed 6 hr/day, 5 days a week, for 28 days to tetramethoxysilane (TMOS) at concentrations of 0, 1, 5, and 10 ppm (Phase I study) and to 0, 15, 30, and 45 ppm (Phase II study). All of the rats exposed to 45 ppm TMOS died or were sacrificed in a moribund state during the 28-day study period. Statistically significant changes were observed in food consumption, body weights, and clinical chemistry parameters in the animals exposed to 30 ppm TMOS. Males exposed to 15 ppm TMOS showed a significant decrease in total protein. No effects were seen in rats exposed to 1, 5, and 10 ppm TMOS. Histopathological lesions related to TMOS exposure were observed in the respiratory tract tissues and eyes of rats exposed to 15, 30, and 45 ppm TMOS. The principal types of lesions observed were ulceration, inflammation, and necrosis of epithelium. At 45 ppm, changes at these sites were severe and present in all animals. Changes at 30 ppm, while occurring in all rats, were much less severe than those seen at 45 ppm. At 15 ppm, the changes were minimal and occurred only in three males and five females. The data of this study showed that TMOS has a steep dose-response curve with no observable effects at 10 ppm, very minimal effects at 15 ppm, moderate to severe effects at 30 ppm, and severe effects and lethality at 45 ppm.

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.

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.

Cutaneous manifestations of early human immunodeficiency virus exposure.

After demonstrating antibodies to the human immunodeficiency virus (HIV), two hundred patients were interviewed and given a complete cutaneous and mucous membrane examination. By means of the Walter Reed Staging Classification System for HIV infection, 155 patients were classified as having Walter Reed stage 1A-2A (WR1A-WR2A) infection. The prevalence of seborrheic dermatitis in this group was 36%. There were no other significant cutaneous findings in the WR1A-WR2A patient population.

Treatment of angiolymphoid hyperplasia of the external ear with carbon dioxide laser.

Two women with long histories of angiolymphoid hyperplasia involving the left external ear were successfully treated with carbon dioxide laser vaporization. In one of these patients a previous treatment with argon laser photocoagulation had proved unsuccessful. Carbon dioxide laser vaporization can be advantageous in the treatment of angiolymphoid hyperplasia, especially when the patient is not a good candidate for cold steel surgery or when the disease process involves confined or convoluted structures such as those on the external ear.

Treatment of dermatofibrosarcoma protuberans with Mohs micrographic surgery.

Ten patients with dermatofibrosarcoma protuberans, all of whom had been treated previously by conventional excisional surgery without success, were treated with Mohs micrographic surgery. A team approach utilizing margin control by Mohs-trained physicians and reconstruction by surgical specialists was employed. Average follow-up exceeds 3 1/2 years, with no recurrences. Microscopically controlled excision appears to be the treatment of choice for dermatofibrosarcoma protuberans.