Real-time PCR检测麻风患者治疗前后及少菌型石蜡标本中麻风菌DNA的价值

目的应用已建立的实时定量荧光PCR(real-time PCR)方法检测少菌型麻风(PB)患者的石蜡标本,探讨该方法对BI为0患者的辅助诊断价值。方法从石蜡标本中提取的DNA进行麻风菌重复序列的特异性real-time PCR检测,包括短程联合化疗(MDT)治疗前后标本共92例;2008年和2013年收集的界限类偏瘤型麻风(BL)石蜡标本分别为7例和8例。并检测70例临床诊断PB和未定类麻风,同时将RT-PCR检测结果与病理分析结果进行比较。结果 92例(LL:19例;BL:20例;BB:20例;BT:16例;TT:17例)不同型别麻风患者的石蜡标本检测阳性率治疗前、治疗后分别为100.00%,100.00%,90.00%,87.50%,50.00%与40.00%,40.00%,0,0,22.22%。比较保存5年前后15例BL患者石蜡标本,检测结果差异无统计学意义。70例临床诊断PB和未定类麻风病患者的石蜡标本real-time PCR检测阳性率为71.43%(50/70),病理检测与临床符合率为51.43%(36/70),两者的阳性检测率差异有统计学意义(P0.05)。结论建立Taq Man(水解探针)技术的real-time PCR方法可以快速、特异、灵敏的检测石蜡标本中的麻风菌DNA,提高抗酸染色阴性标本的检测阳性率,对临床诊断的指导意义重大。     

石蜡包埋组织中麻风菌基因扩增试验初探

目的建立用石蜡包埋组织中麻风菌DNA进行基因扩增的试验.方法用Texpat试盒将石蜡切片脱蜡、破壁和释放麻风菌DNA并用100%乙醇沉淀纯化DNA,用引物RPOT(F)和RPUT (R)进行PCR.结果细菌指数大于1+的33份石蜡包埋标本,28份PCR阳性,细菌指数为0的标本PCR阴性.阴性对照呈阴性反应,阳性对照呈阳性反应,方法敏感度达0.04pgDNA水平.结论用本法可从福尔马林固定的石蜡包埋组织切片中释放麻风菌DNA,并获得满意基因扩增结果.     

麻风杆菌PCR检测方法的优化及应用

目的:研建一种适用于临床中检测麻风杆菌(ML)的分子生物学方法,并优化检测条件以提高检测方法的灵敏度。方法:用麻风杆菌纯DNA作模板,对PCR反应体系中模板浓度、引物浓度等反应条件进行优化,以建立高敏感性的PCR反应体系,并与实时荧光定量PCR(Real-time PCR)方法比较,对麻风病人标本进行检测,确定优化后两法的反应体系及两法的临床检测意义。结果:经过大量的实验比对表明,反应体系引物含量为0.2μL×100μmol时,模板浓度10-1条ML/m L可以作为稳定的可检测到条带的模板浓度的下限。对52例各型麻风患者标本的检测结果显示常规PCR检测阳性数(49/52)略高于Real-time PCR(47/52),但Real-time PCR操作程序简单和需时短,且成本低。结论:通过对PCR反应条件中DNA模板浓度和引物浓度的优选,提高了PCR检测麻风杆菌DNA的灵敏度;对于不同PCR方法的选择方面,Real-time PCR比常规PCR简捷快速,而常规PCR灵敏度略高于Realtime PCR。     

建立巢式PCR和异源双链法检测石蜡标本麻风菌DDS耐药株

目的：为了能检出石蜡包埋组织中麻风菌氨苯砜（DDS）的耐药基因folPl，有必要建立敏感、特异的巢式PCR，为开展回顾性麻风菌DDS耐药流行病学研究服务。方法：从石蜡组织中提取麻风菌DNA，建立巢式PCR，优化PCR条件，扩增DDS耐药基因片段。用异源双链法筛选突变菌株，并经直接测序进一步证实。结果：巢式PCR将PCR检测folPl的敏感性，从6．5％（3／46）提高到76．7％（33／43）；优化后的巢式PCR，最终使敏感性更进一步提高至90．7％（39／43）。8株folPl突变菌、两种突变型被发现。结论：巢式PCR是一项快速、简便、可行的方法。通过优化多项PCR条件，提高了石蜡包埋组织中folPl检测的敏感性和特异性。异源双链法可用于筛选DDS耐药突变菌株。     

单管巢式实时PCR检测麻风杆菌方法的建立及初步应用

目的:明确单管巢式实时PCR检测麻风杆菌的特异性、灵敏性和重复性。方法:以麻风杆菌hsp18基因为靶基因设计引物和探针,建立单管巢式实时PCR方法检测麻风杆菌并与抗酸染色及普通PCR进行特异性和灵敏性的比较。结果:单管巢式实时PCR检测下限为2.1fg质粒DNA,普通PCR为21fg质粒DNA,敏感性高10倍;与8种非麻风杆菌无交叉反应;批内相对标准偏差(RSD)为0.24%~1.08%,批间RSD为0.09%~2.8%。对比检测54例麻风疑似标本中,抗酸染色、普通PCR和单管巢式实时PCR的敏感性分别为81.3%(26/32)、87.5%(28/32)和96.9%(31/32)。结论:单管巢式实时PCR特异性强、敏感性高且重复性好。     

皮肤分枝杆菌DNA提取方法比较分析

目的 比较分枝杆菌DNA提取的常用方法。 方法 分别采用传统的冻融法和两种试剂盒（A、B）对不同浓度结核分枝杆菌、麻风分枝杆菌和耻垢分枝杆菌纯菌悬液及模拟标本进行总DNA提取。通过检测DNA纯度和PCR产物比较3种方法的提取效果,并应用临床皮肤组织标本进一步验证。 结果 3种方法提取的分枝杆菌DNA均能用于PCR检测。试剂盒A获得的DNA纯度最高,冻融法其次,试剂盒B杂质最多;灵敏性检测显示3种方法可检测出的纯菌悬液最低浓度皆为102菌细胞/ml;对于模拟标本,在103菌细胞/ml时检出率为100%,在102菌细胞/ml时试剂盒A、B和冻融法检出率分别为60%（12/20）、55%（11/20）、55%（11/20）;临床标本检测结果提示,试剂盒B可用于石蜡标本DNA的提取,对分枝杆菌感染组织提取率同其它两种方法基本一致。 结论 试剂盒A通过多次过柱洗涤可快速获得分枝杆菌的优质基因组DNA,为实验研究及临床检测的首选;试剂盒B单次试剂处理除适用于石蜡标本提取DNA外,可用于新鲜组织标本的协同检测。     

巢式PCR检测石蜡包埋组织中着色霉、孢子丝菌和马尔尼菲青霉

目的 评价巢式PCR对不同深部真菌感染组织石蜡包埋标本检测的价值。方法 收集着色芽生菌病、孢子丝菌病、马尔尼菲青霉病及其他深部真菌病石蜡组织标本共44份,行组织病理观察并提取石蜡组织标本中DNA。使用针对着色霉、孢子丝菌及马尔尼菲青霉核糖体DNA特定区域的特异性巢式PCR引物,分别对所提取的真菌DNA进行扩增。分析巢式PCR对这3种病原真菌扩增的敏感性和特异性,并与组织病理检查方法比较。结果 20例着色芽生菌病组织蜡块中8例扩增阳性,10例孢子丝菌病组织蜡块中7例扩增阳性,10例马尔尼菲青霉病组织蜡块均扩增阳性,其余对照深部真菌病组织蜡块扩增均为阴性,巢式PCR检测3种真菌的敏感性分别为40%、70%和100%,特异性均达到100%。组织病理检查3种真菌的阳性率分别为95%、70%、80%。结论 巢式PCR扩增石蜡包埋组织中的真菌DNA是诊断深部真菌病的一种方法,尤其适用于诊断马尔尼菲青霉病。     

以S2-R2为引物PCR检测石蜡切片中的孢子丝菌

目的 探讨应用特异性引物PCR方法检测石蜡切片中孢子丝菌的可行性。方法 选取30份大连及10份长春地区临床疑诊孢子丝菌病的石蜡切片标本,采用改良的微波脱蜡、液氮研磨-CTAB破壁法提取DNA,以特异性引物S2-R2进行PCR扩增,与真菌培养结果进行比对。结果 30份大连地区标本中22份见阳性PCR扩增产物（73.33%）。真菌培养阳性的22份标本中20份PCR出现阳性扩增产物（91%）,真菌培养阴性的8份标本中2 份PCR出现阳性扩增产物。10份长春地区标本7份见阳性PCR扩增产物（70%）。结论 以S2-R2为引物的 PCR适用于孢子丝菌病石蜡切片中病原菌的检测。     

PCR分析在监测瘤型麻风病人化疗效果中的评价

作者报告用多聚酶链式反应(PCR)分析技术研究动物实验化疗后及瘤型麻风病人疗后的皮肤活检标本中的麻风菌的结果。将1×10~7条麻风菌接种于含不同浓度药物的BACTEC培养基小瓶中,当观察到麻风菌代谢活性为0时,从各小瓶中取出1×10~5条麻风菌,经提纯、溶酶消化后,以氯仿-异戊醇提取DNA,进行PCR分析。实验化疗系用含菌10~9/足垫的裸鼠,经含利福平或氨苯砜或MDT的饲料喂食12个月时,     

麻风菌基因扩增试验条件的优化及其影响因素

通过模板DNA制备方法和循环参数的优化,使PCR的敏感性从20条菌/μ1提高到0.2条菌/μ1(100倍)。对16份鼠足垫标本(其中5份镜检阳性,11份阴性),26份麻风病人活检组织(19份镜检阳性,7份阴性)和刮取液(镜检19份阳性,7份阴性)进行检测并分析了对PCR的影响。讨论了该方法在麻风菌检测和鉴定中的应用前景。     

Outcomes and adverse effects of ablative vs nonablative lasers for skin resurfacing: A systematic review of 1093 patients

It is generally believed that ablative laser therapies result in prolonged healing and greater adverse events when compared with nonablative lasers for skin resurfacing. To evaluate the efficacy of ablative laser use for skin resurfacing and adverse events as a consequence of treatment in comparison to other modalities, a PRISMA‐compliant systematic review (Systematic Review Registration Number: 204016) of twelve electronic databases was conducted for the terms “ablative laser” and “skin resurfacing” from March 2002 until July 2020. Studies included meta‐analyses, randomized control trials, cohort studies, and case reports to facilitate evaluation of the data. All articles were evaluated for bias. The search strategy produced 34 studies. Of 1093 patients included in the studies of interest, adverse events were reported in a total of 106 patients (9.7%). Higher rates of adverse events were described in nonablative therapies (12.2% ± 2.19%, 31 events) when compared with ablative therapy (8.28% ± 2.46%, 81 events). 147 patients (13.4%) reported no side effects, 68 (6.22%) reported expected, transient self‐resolving events, and five (0.046%) presented with hypertrophic scarring. Excluding transient events, ablative lasers had fewer complications overall when compared with nonablative lasers (2.56% ± 2.19% vs 7.48% ± 3.29%). This systematic review suggests ablative laser use for skin resurfacing is a safe and effective modality to treat a range of pathologies from photodamage and acne scars to hidradenitis suppurativa and posttraumatic scarring from basal cell carcinoma excision. Further studies are needed, but these results suggest that ablative lasers are a superior, safe, and effective modality to treat damaged skin.     

New fillers for the new man

ABSTRACT:  Two new collagen-based lidocaine-containing dermal fillers, ArteSense™/ArteFill™ (Artes Medical, San Diego, CA) and Evolence® (Colbar LifeScience Ltd., Herzliya, Israel), have proved to be of particular interest to men, many of whom seek a long-lasting or permanent correction. ArteFill™ has been available in the United States since 2006, and it is expected that Evolence® will reach the American market in 2008. The properties of the two products will be described, and experience based on the administration of many hundreds of syringes of both products by a Canadian dermatologist will be detailed here, with tips and precautions to optimize patient outcomes.     

Deutliche Einschränkung der Lebensqualität bei Patienten mit Pemphigus vulgaris: Ergebnisse der deutschen Bullous Skin Disease (BSD)‐Studiengruppe

Background: Pemphigus vulgaris is a potentially life‐threatening autoimmune disorder of the skin and mucous membranes characterized by antibodies against epidermal adhesion molecules. Clinically characteristic are painful chronic blisters or erosions of mucous membranes and skin. There are no published studies on the impact o this disease on quality of life. Patients and methods: This registration was performed within the scope of the German BSD (Bullous Skin Disease) study group, from November 1997 until January 2002. A total of 36 patients with the first diagnosis of pemphigus vulgaris were registered at the university hospitals of Dresden, Erlangen, Kiel, Mannheim, München and Würzburg. Thirty of the 36 (83 %) patients participated in the quality of life questionnaire utilizing the German version of ‘Dermatology Life Quality Index’ (DLQI) provided by A. Y. Finlay. The DLQI varies from 0 to 30 with an increased DLQI score indicating a decrease in quality of quality. Results: The overall DLQI total score of 10 ± 6,7 in the investigated pemphigus patients was significantly increased in comparison to other skin diseases. Conclusions: These results suggest that the DLQI can be a very useful additional outcome criteria for clinical studies with pemphigus vulgaris and in the treatment of these patients.     

Genetic alterations in RAS‐regulated pathway in acral lentiginous melanoma

Studies integrating clinicopathological and genetic features have revealed distinct patterns of genomic aberrations in Melanoma. Distributions of BRAF or NRAS mutations and gains of several oncogenes differ among melanoma subgroups, while 9p21 deletions are found in all melanoma subtypes. In the study, status of genes involved in cell cycle progression and apoptosis was evaluated in a panel of 17 frozen primary acral melanomas. NRAS mutations were found in 17% of the tumors. In contrast, BRAF mutations were not found. Gains of AURKA gene (20q13.3) were detected in 37.5% of samples, gains of CCND1 gene (11q13) or TERT gene (5p15.33) in 31.2% and gains of NRAS gene (1p13.2) in 25%. Alterations in 9p21 were identified in 69% of tumors. Gains of 11q13 and 20q13 were mutually exclusive, and 1p13.2 gain was associated with 5p15.33. Our findings showed that alterations in RAS‐related pathways are present in 87.5% of acral lentiginous melanomas.     

Mutations in mevalonate pathway genes in patients with familial or sporadic porokeratosis

Porokeratosis comprises heterogeneous keratinization disorders that are characterized by one or more atrophic patches surrounded by a ridge‐like cornoid lamella. In this study, we evaluated seven families affected by porokeratosis and five sporadic patients of the disease in a Chinese population. We performed Sanger sequencing of exons and flanking intron–exon boundaries of mevalonate pathway genes (MVD, MVK, PMVK and FDPS) and of SLC17A9. In five familial and three sporadic patients, we detected six variations, including four novel mutations (MVD c.1A>G; p.Met1?, c.916G>A; p.Ala306Thr, c.1013+1G>A, and PMVK c.65A>G; p.Lys22Arg) and two recurrent mutations (MVD c.746T>C; p.Phe249Ser, and MVK c.1028T>C; p.Leu343Pro). We then applied I‐TASSER and iGEMDOCK to assess these variants for probable functional impacts. The findings of this study extend the mutation spectrum of porokeratosis and provide further evidence for the genetic basis of this disease.     

Occupational allergic contact dermatitis caused by decorative plants

12 cases of occupational allergic contact dermatitis caused by decorative plants were diagnosed in a 14-year period. The patients were middle-aged, and their average exposure time was 13 years. The plant families and plants causing occupational contact dermatitis were Compositae (5 patients: chrysanthemum, elecampane, gerbera, feverfew), Alstroemeriaceae (5 patients, Alstroemeria ), Liliaceae (4 patients; tulip, hyacinth). Amaryllidaceae (2 patients: narcissus) and Caryophyllaceae (2 patients; carnation, cauzeflower). The known chemical allergens causing dermatitis were tuliposide-A and sesquiterepene lactones, such as alantolactones and parthenolide, in the Liliaceae and Compositae families. 7 of the 12 patients were able to continue their work; 5 were not because of severe relapses of skin symptoms. The plant allergen and extract series currently available are of great help in the diagnosis.