In this study, the technical performance of culture, two commercially available polymerase chain reaction (PCR) tests, rapid plate agglutination (RPA) test, hemagglutination inhibition (HI) test, and ...eight commercially available enzyme-linked immunosorbent assays (ELISAs) were compared for the detection of avian mycoplasma infections from 3 days postinfection (d.p.i.) through 35 d.p.i. The tests were carried out on samples from specified pathogen-free layers that were infected at 66 wk of age with recent Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) field strains, MS and MG ATCC strains, and Mycoplasma imitans (MIM), respectively. Results showed a high percentage of positive samples in the homologous infected groups and a high percentage of negative samples (100%) in the uninfected and heterologous infected groups during 35 d.p.i. of both culture and PCR tests. For the group infected with the MG 15302 ATCC strain, serology was more sensitive than bacteriology. All MG and MS tests, with the exception of MG ELISA kit D showed a lower percentage of positive samples during 35 d.p.i. for the detection of the MG and MS ATCC strain infection compared with that of the field strains. Also, the number of cross-reactions (false positives) in the serologic tests was lower after infection with an ATCC strain than after an infection with the MG or MS field strain. Contradictory to other studies, the ELISAs and the RPA test using undiluted serum showed a relatively high number of false-positive results. The MG ELISAs (except ELISA kit D) showed more false-positive results (up to 37%) in the MIM-infected group than in the MS-infected groups. This was not unexpected, as MIM and MG have a close antigenic relationship. The results of the serologic tests in this study showed that a certain level of false-positive results can be expected in about any serologic test. Although the level of false-positive results varied between several serologic tests, this study showed that it is not advisable to rely completely on one test (system) only.
The aim of this study was to evaluate the sensitivity and the detection limit of a real-time polymerase chain reaction (Q-PCR) developed for the qualitative and quantitative detection of Mycoplasma ...gallisepticum. No cross-reactivity was observed with DNA from other important avian mycoplasmas, including Mycoplasma synoviae and Mycoplasma meleagridis. However, the Q-PCR could not distinguish between M. gallisepticum and Mycoplasma imitans. The Q-PCR had detection limits 10 to 1000 times lower than a conventional commercial PCR method and than culture. The Q-PCR was used quantitatively by incorporating a set of external M. gallisepticum DNA standards, derived from a M. gallisepticum log-phase culture of a known concentration. The number of colony-forming unit equivalents per millilitre in tracheal swabs from experimentally infected birds could be determined from a single sample. The method had good reproducibility and correlated well with standard counting techniques using culture. It can be concluded that the Q-PCR described is suitable for qualitative and quantitative detection of M. gallisepticum in clinical samples.
PCR en temps réel pour la détection qualitative et quantitative de Mycoplasma gallisepticum
Le but de cette étude a été d'évaluer la sensibilité et les limites de détection du test d'amplification en chaîne par polymérase en temps réel (Q-PCR) développé pour la détection qualitative et quantitative de Mycoplasma gallisepticum (Mg). Aucune réaction croisée n'a été observée avec l'ADN d'autres mycoplasmes aviaires importants, incluant M. synoviae et M. meleagridis . Cependant, le test Q-PCR n'a pas pu différencier Mg de M. imitans. Les limites de détection ont été de 10 à 1000 fois inférieures à celles d'un test PCR du commerce (cPCR) et à la culture. Le test Q-PCR a été utilisé quantitativement en incorporant un set de standards externes d'ADN de Mg dérivé d'une culture de Mg, en phase logarithmique, de concentration connue. Le nombre d'équivalents unité formant colonie/ml dans les écouvillons trachéaux des animaux infectés expérimentalement a pu être déterminé à partir d'un seul échantillon. La reproductibilité de la méthode a été bonne et une bonne corrélation a été observée avec les techniques standard de comptage après culture. Il peut être conclu que le test Q-PCR décrit est approprié à la détection qualitative et quantitative de Mg à partir d'échantillons cliniques.
Real-Time-PCR für den qualitativen und quantitativen Nachweis von Mycoplasma gallisepticum
Ziel dieser Studie war es, die Sensibilität und die Nachweisgrenze einer für die qualitative und quantitative Bestimmung von Mycoplasma gallisepticum (MG) entwickelten Real-Time-Polymerasekettenreaktion (Q-PCR) zu untersuchen. Mit der DNS anderer wichtiger aviärer Mykoplasmen einschließlich M. synoviae und M. meleagridis wurden keine Kreuzreaktionen beobachtet. Die Q-PCR konnte jedoch nicht zwischen MG und M. imitans unterscheiden. Die Q-PCR hatte eine 10-1000-mal niedrigere Nachweisgrenze als die konventionelle kommerzielle PCR (cPCR) und die kulturelle Nachweismethode. Die Q-PCR wurde zum quantitativen Nachweis genutzt, indem ein Satz externer MG-DNS-Standards aus MG-log-Phasen-Kulturen mit bekannter Konzentration inkorporiert wurde. Die Zahl der Kolonie bildenden Einheiten/ml in Trachealtupfern von experimentell infizierten Tieren konnte in einer einzigen Probe bestimmt werden. Die Methode weist eine gute Reproduzierbarkeit auf und korrelierte mit Standard-Zählverfahren bei der Kultivierungsmethode. Daraus kann geschlossen werden, dass die beschriebene Q-PCR für den qualitativen und quantitativen Nachweis von MG in Untersuchungsproben geeignet ist.
PCR a tiempo real par la detección cuantitativa y cualitativa de Mycoplasma gallisepticum
El objetivo de este estudio fue el de evaluar la sensibilidad y el límite de detección de una reacción en cadena de la polimerasa a tiempo real (Q-PCR) desarrollado para la detección cualitativa y cuantitativa de Mycoplasma gallisepticum (Mg). No se observó reactividad cruzada con el ADN de otros importantes micoplasmas aviares, incluidos M. synoviae y M. meleagridis. Aún así, la Q-PCR no pudo distinguir entre Mg y M. imitans. LA Q-PCR presentó límites de detección de 10 a 1,000 veces menores que la PCR comercial convencional (cPCR) y que el cultivo. La Q-PCR se utilizó como método cuantitativo mediante la incorporación de un grupo de ADN de Mg estándares externos, derivados de un cultivo de Mg en fase log, de concentración conocida. El número de de equivalentes de unidades formadoras de colonias/ml en hisopos traqueales de aves infectadas experimentalmente pudo ser determinado a partir de una sola muestra. El método presentó buena reproducibilidad y se correlacionó bien con las técnicas estándares de contaje usadas en cultivos. Se puede concluir que la Q-PCR descrita es útil para la detección cualitativa y cuantitativa de Mg en muestras clínicas.
Hidradenitis suppurativa (HS) is a chronic, inflammatory, recurrent, debilitating skin disease. Several treatment modalities are available, but most of them lack high-quality evidence. A systematic ...search was performed to identify all randomized controlled trials for the treatment of HS in order to review and evaluate the evidence. Recommendations for future randomized controlled trials include using validated scores, inclusion of patient rated outcomes, and thorough report of side effects. Evidence for long-term treatment and benefit/risk ratio of available treatment modalities is needed in order to enhance evidence-based treatment in daily clinical practice. Combining surgery with antiinflammatory treatment warrants further investigation.
Summary
Background Hidradenitis suppurativa is a chronic inflammatory skin disease characterized by abscess formation, predominantly in the axillae and groins. The disease is difficult to treat and ...has a severe impact on quality of life. Recently, several case reports have been published describing successful treatment of hidradenitis suppurativa with infliximab and other tumour necrosis factor α inhibitors.
Objectives To evaluate the long‐term efficacy of a single course of infliximab.
Methods Ten patients with severe, recalcitrant hidradenitis were treated with infliximab (three infusions of 5 mg kg−1 at weeks 0, 2 and 6) and followed up for at least 1 year. The disease activity was measured using laboratory parameters and a recently developed acne score. The patients rated the efficacy of infliximab on a 10‐point scale at regular intervals. Quality of life was measured before and after treatment using the Dermatology Quality of Life Index (DQLI).
Results All patients improved within 2–6 weeks. The average acne score diminished from 164 ± 50 (mean ± SD) before treatment to 89 ± 49 after 1 year (P = 0·002). The mean CRP (C‐reactive protein) was reduced from 31·7 mg mL−1 to 5·5 mg mL−1 after 1 month (P = 0·015). Patients judged the efficacy with a score of 7·9. The mean DQLI was reduced from 18·4 ± 7·9 before treatment to 9·3 ± 9·1 after 1 year (P = 0·007). In three patients long‐lasting improvement was observed, with no recurrence of lesions in a 2‐year follow‐up period. The other patients showed recurrence of lesions after 8·5 months (range 4·3–13·4 months).
Conclusions Infliximab is an effective treatment in severe hidradenitis suppurativa, leading to reduction of symptoms for a prolonged period.
Summary Chronic ulceration of the lower leg is a frequent condition, with a prevalence of 3–5% in the population over 65 years of age. The incidence of ulceration is rising as a result of the ageing ...population and increased risk factors for atherosclerotic occlusion such as smoking, obesity and diabetes. Ulcers can be defined as wounds with a ‘full thickness depth’ and a ‘slow healing tendency’. In general, the slow healing tendency is not simply explained by depth and size, but caused by an underlying pathogenetic factor that needs to be removed to induce healing. The main causes are venous valve insufficiency, lower extremity arterial disease and diabetes. Less frequent conditions are infection, vasculitis, skin malignancies and ulcerating skin diseases such as pyoderma gangrenosum. But even rarer conditions exist, such as the recently discovered combination of vasculitis and hypercoagulability. For a proper treatment of patients with leg ulcers, it is important to be aware of the large differential diagnosis of leg ulceration.
In diabetic patients, wound healing is impaired. We studied the pathogenesis behind this clinical observation by characterizing the pattern of deposition of extracellular matrix (ECM) molecules and ...the cellular infiltrate in chronic (>8 wk) diabetic wounds, compared with chronic venous ulcers and an acute wound healing model. Punch biopsies were obtained from the chronic ulcer margins and control samples were collected from upper leg skin 5, 19, 28 d and 12 and 18 mo postwounding (p.w.). T cells, B cells, plasma cells, granulocytes and macrophages, and the ECM molecules fibronectin (FN), chondroitin sulfate (CS), and tenascin (TN) were visualized using immunohistochemical techniques. Expression of FN, CS, and TN was detected in dermal tissue early in normal wound healing (5–19 d p.w.). Abundant staining was seen 3 mo p.w., returning to prewounding levels after 12–18 mo p.w. In the dermis of chronic diabetic and venous ulcers with a duration of 12 mo or more, a prolonged presence of these ECM molecules was noted. Compared with normal wound healing: (i) the CD4/CD8 ratio in chronic wounds was significantly lower (p < 0.0027) due to a relatively lower number of CD4+ T cells; (ii) a significantly higher number of macrophages was present in the edge of both type of chronic ulcers (p < 0.001 versus day 29 p.w.); and (iii) more B cells and plasma cells were detected in both type of chronic wounds compared with any day in the acute wound healing model (p < 0.04 for CD20+ and p < 0.01 for CD79a+ cells). These data indicate that important differences exist in the cellular infiltrate and ECM expression patterns of acute, healing versus chronic wounds, which may be related to the nonhealing status of chronic wounds.
Summary
Infliximab (Remicade®; Schering‐Plough, Kenilworth, NJ, U.S.A.) is a chimeric monoclonal antibody that acts as a tumour necrosis factor‐α inhibitor. Infliximab is registered for the treatment ...of rheumatoid arthritis, psoriatic arthritis, Crohn disease, ulcerative colitis, ankylosing spondylitis and plaque‐type psoriasis. Like other foreign protein‐derived agents, infliximab may lead to infusion reactions during and after infusion. Infusion reactions occur in 3–22% of patients with psoriasis treated with infliximab. Most of these reactions are mild or moderate and only few are severe. Nevertheless, they may lead to discontinuation of treatment. As infliximab for psoriasis is prescribed as a last resort and is in most cases very effective, discontinuation of treatment is undesirable. With proper care and prevention of the infusion reactions the need to discontinue treatment with infliximab can be diminished. The objective of this article is to present a guideline for the management of infliximab‐related infusion reactions, based on the best available evidence. This guideline can be used in patients with psoriasis as well as in dermatology patients receiving infliximab for off‐label indications such as hidradenitis suppurativa or pyoderma gangrenosum.
Background Episodes of microvascular proliferation associated with volume expansion have been observed in arteriovenous malformations (AVMs) of skin and soft tissue. Objective We sought to ...investigate the relationship between a microvascular proliferative response and flow velocity in AVMs. Methods Resection specimens of 80 AVMs were clinically categorized as either high- or low-flow lesions, and histopathologically screened for the presence of microvessels, inflammation, thrombosis, or a combination of these. Immunohistochemistry was performed with endoglin (CD105), von Willebrand factor, and fibrinogen antibodies. Results Clinically, 37 AVMs were classified as high-flow lesions and 43 as low-flow lesions. In 81% of high-flow lesions microvascular proliferations were seen versus in 14% of low-flow lesions ( P < .005). In high-flow lesions, which were embolized before surgery (30% of all), 88% showed microvascular proliferation, 88% inflammation, and 33% thrombosis. However, similar vasoproliferative responses were also observed in nonembolized AVM (69% high-flow and 14% low-flow lesions). Endoglin was more frequently expressed in high-flow lesions. Extracellular von Willebrand factor staining was found in most lesions, irrespective of flow type or presence of microvascular proliferations. Limitations The study was carried out at a single tertiary referral center. Conclusions Microvascular proliferative masses in AVMs appear to be strongly associated with high-flow characteristics. This could be explained to some extent by previous therapeutic embolization and/or inflammation in the lesion. However, occurrence of similar microvascular responses in AVM that were not embolized before surgery suggests that the biomechanical effects of high flow in these lesions may also have an angiogenic effect.
Before interventions to control horizontal transmission of Mycoplasma gallisepticum can be tested, a suitable experimental model should be available. Transmission dynamics in a flock can be ...quantified by two parameters: the average number of secondary cases infected by one typical infectious case ( R(0)) and the number of new infections that occur due to one infectious animal per unit of time (β). The transmission dynamics of M. gallisepticum have not been studied experimentally, so the aim of this study was to examine the horizontal transmission of M. gallisepticum . The study was carried out using a pairwise design with three different inoculation doses. Every pair consisted of an inoculated chicken and a susceptible in-contact chicken. Five susceptible individually housed chickens were placed in between pairs in order to measure airborne transmission. Infection was detected by serology, quantitative polymerase chain reaction and culture. The inoculated and in-contact chickens were equally infectious and the pairs could be regarded as independent. The R(0) was estimated to be greater than 1 (...; 95% confidence interval, 4.5 to ...), the estimated β was 0.22 per day and there was no significant difference between the different inoculation doses. It was concluded that the animal model as described in this study meets the conditions for the establishment of transmission dynamics of M. gallisepticum and therefore can be used to establish the quantitative effect of intervention measures on horizontal transmission.
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