淋球菌性菌血症的快速筛查及鉴定

目的探讨16S rDNA检测技术在快速筛查鉴定淋球菌性菌血症中的应用价值.方法选择1例反复规律性发热、畏寒1个月入院的41岁男性菌血症患者,入院前几天开始出现尿急、尿频和尿痛症状,大便正常,贫血消瘦外容,皮肤黏膜稍苍白,无出血点及皮疹,全身浅表淋巴结未及肿大,血尿常规异常,肝肾功能尚可.采用PCR检测菌血症患者外周血病原菌16S rDNA,对其扩增产物进行DNA测序,再将DNA序列结果与GenBank中的已知序列进行同源性比较;同时对外周血病原菌进行分离培养鉴定及药敏实验.结果16S rDNA扩增产物序列与GenBank中的已知序列进行同源性比较后确定为淋病奈瑟菌,同外周血分离培养鉴定结果一致.结论检测淋球菌性菌血症患者外周血16S rDNA具有高效率、高特异性、高灵敏度、快速准确等特点.     

淋球菌性菌血症的快速筛查及鉴定

目的探讨16S rDNA检测技术在快速筛查鉴定淋球菌性菌血症中的应用价值.方法选择1例反复规律性发热、畏寒1个月入院的41岁男性菌血症患者,入院前几天开始出现尿急、尿频和尿痛症状,大便正常,贫血消瘦外容,皮肤黏膜稍苍白,无出血点及皮疹,全身浅表淋巴结未及肿大,血尿常规异常,肝肾功能尚可.采用PCR检测菌血症患者外周血病原菌16S rDNA,对其扩增产物进行DNA测序,再将DNA序列结果与GenBank中的已知序列进行同源性比较;同时对外周血病原菌进行分离培养鉴定及药敏实验.结果16S rDNA扩增产物序列与GenBank中的已知序列进行同源性比较后确定为淋病奈瑟菌,同外周血分离培养鉴定结果一致.结论检测淋球菌性菌血症患者外周血16S rDNA具有高效率、高特异性、高灵敏度、快速准确等特点.     

淋球菌性菌血症的快速筛查及鉴定

目的探讨16S rDNA检测技术在快速筛查鉴定淋球菌性菌血症中的应用价值.方法选择1例反复规律性发热、畏寒1个月入院的41岁男性菌血症患者,入院前几天开始出现尿急、尿频和尿痛症状,大便正常,贫血消瘦外容,皮肤黏膜稍苍白,无出血点及皮疹,全身浅表淋巴结未及肿大,血尿常规异常,肝肾功能尚可.采用PCR检测菌血症患者外周血病原菌16S rDNA,对其扩增产物进行DNA测序,再将DNA序列结果与GenBank中的已知序列进行同源性比较;同时对外周血病原菌进行分离培养鉴定及药敏实验.结果16S rDNA扩增产物序列与GenBank中的已知序列进行同源性比较后确定为淋病奈瑟菌,同外周血分离培养鉴定结果一致.结论检测淋球菌性菌血症患者外周血16S rDNA具有高效率、高特异性、高灵敏度、快速准确等特点.     

Human β-defensin-2 and skin diseases

Human β-defensin-2 (hBD-2) is an endogenous antimicrobial peptide recently found in the skin with broad-spectrum antimicrobial activity and unique mechanism of function. Recent studies have confirmed that the expression of hBD-2 is upregulated in the lesions of some dermatoses, such as psoriasis,acne vulgaris, verruca, dermatophytosis and basal cell carcinoma, etc. The susceptibility to bacterial and viral infections may be associated with the decreased or absent expression of hBD-2. Further researches into HBD-2will provide a new direction for the prevention and treatment of skin diseases.     

梅毒非特异性类脂质反应素抗体试验操作的改进及应用评价

Objective To estimate the application value of a standard operating procedure (SOP) in the detection of syphilitic anticardiolipin reagin. Methods Clinical laboratories from 9 local hospitals in Shanghai participated the program. Quality control samples with unknown target value were qualitatively and quantitatively examined according to the uniform SOP in these laboratories with the same reagent and facility of horizontal reaction. External quality assessment (EQA) was carried out by using seven serum samples with no, or low (1∶ 128 dilution) to high (1∶1 dilution) concentrations of target before and after the implementation of SOP. The test results were statistically analyzed and the reasons for the detecting error were assessed. Results A total of 388 tests were performed in the 9 clinical laboratories. The total accuracy rate was 93.0%, including 40.2% in the detection of samples with 1 ∶ 8 dilution of target, 49.2% in the detection of samples with 1 ∶ 16 dilution of target, and 3.6% in the detection of samples with 1 ∶ 32 dilution of target. No forward bias was observed in these tests. There was a significant difference in the accuracy rate between the two times of EQA before and after the implementation of SOP (x2 = 4.17, P < 0.05). Conclusions The improved standard procedure for nontreponemal antigen test is beneficial to the decrease of testing error, and may provide a basis for the establishment of SOP and implementation of internal quality assessment.     

梅毒非特异性类脂质反应素抗体试验操作的改进及应用评价

Objective To estimate the application value of a standard operating procedure (SOP) in the detection of syphilitic anticardiolipin reagin. Methods Clinical laboratories from 9 local hospitals in Shanghai participated the program. Quality control samples with unknown target value were qualitatively and quantitatively examined according to the uniform SOP in these laboratories with the same reagent and facility of horizontal reaction. External quality assessment (EQA) was carried out by using seven serum samples with no, or low (1∶ 128 dilution) to high (1∶1 dilution) concentrations of target before and after the implementation of SOP. The test results were statistically analyzed and the reasons for the detecting error were assessed. Results A total of 388 tests were performed in the 9 clinical laboratories. The total accuracy rate was 93.0%, including 40.2% in the detection of samples with 1 ∶ 8 dilution of target, 49.2% in the detection of samples with 1 ∶ 16 dilution of target, and 3.6% in the detection of samples with 1 ∶ 32 dilution of target. No forward bias was observed in these tests. There was a significant difference in the accuracy rate between the two times of EQA before and after the implementation of SOP (x2 = 4.17, P < 0.05). Conclusions The improved standard procedure for nontreponemal antigen test is beneficial to the decrease of testing error, and may provide a basis for the establishment of SOP and implementation of internal quality assessment.     

梅毒非特异性类脂质反应素抗体试验操作的改进及应用评价

Objective To estimate the application value of a standard operating procedure (SOP) in the detection of syphilitic anticardiolipin reagin. Methods Clinical laboratories from 9 local hospitals in Shanghai participated the program. Quality control samples with unknown target value were qualitatively and quantitatively examined according to the uniform SOP in these laboratories with the same reagent and facility of horizontal reaction. External quality assessment (EQA) was carried out by using seven serum samples with no, or low (1∶ 128 dilution) to high (1∶1 dilution) concentrations of target before and after the implementation of SOP. The test results were statistically analyzed and the reasons for the detecting error were assessed. Results A total of 388 tests were performed in the 9 clinical laboratories. The total accuracy rate was 93.0%, including 40.2% in the detection of samples with 1 ∶ 8 dilution of target, 49.2% in the detection of samples with 1 ∶ 16 dilution of target, and 3.6% in the detection of samples with 1 ∶ 32 dilution of target. No forward bias was observed in these tests. There was a significant difference in the accuracy rate between the two times of EQA before and after the implementation of SOP (x2 = 4.17, P < 0.05). Conclusions The improved standard procedure for nontreponemal antigen test is beneficial to the decrease of testing error, and may provide a basis for the establishment of SOP and implementation of internal quality assessment.     

Integrated Skin Microbiome-Metabolome Analysis Reveals Altered Bacterial Community Composition and Metabolites in Psoriasis

Objective: Current theories highlight the role of the microbiome in the pathogenesis of psoriasis. Additionally, abnormal metabolism can alter disease processes in terms of occurrence, progression, and prognosis. Therefore, an integrative microbiome and metabolome analysis of the skin may aid in understanding the disease pathogenesis and identify therapeutic targets for psoriasis.Methods: We recruited 22 patients with psoriasis and 22 age- and sex-matched healthy controls. Skin swabs were collec...     

Visual Clues for the Histopathologic Diagnosis of Psoriasis: A Retrospective Case-Control Study

Objective: The histopathologic diagnosis of psoriasis remains challenging. This study aimed to uncover clues for the histopathologic diagnosis of psoriasis in patients whose lesions required differentiation from other inflammatory skin disorders.Methods: A retrospective analysis was conducted between psoriasis biopsy sections and other inflammatory skin diseases sections. The psoriasis and control groups were compared regarding the pathological characteristics, including the ? sign, hypogranulos...     

SLE患者基因组DNA甲基化水平的检测

Objective To assess the DNA methylation status in peripheral blood mononulclear cells (PBMCs)from patients with systemic lupus erythematosus (SLE) using a constructed methyl-CpG-binding domain (MBD) proteins.Methods A recombinant plasmid MBD-pET30b+ was transformed into E.coli (DH5a)for clonal expansion followed by sequencing.Then,plasmids with correct sequence were transformed into E coli BL21 (DE3) for the expression of MBD proteins under the induction of isopropy-β-D-thiogalactoside (IPTG).The expression products were purified by Ni-NTA chromatography and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)and Western blotting.Cultured 293T cells and isolated PMBCs from 21 patients with SLE and 15 normal human controls were immunostained with the expressed protein,and analyzed by fluorescence microscopy and flow cytometry.Results The sequence of recombinant plasmid was proved to be consistent with the expectation.SDS-PAGE and Western blotting demonstrated that the molecular weight of expressed tetrameric-protein was 46000,with the presence of N-terminal His-tag and C-terminal HA-tag.As fluorescence microscopy showed,the MBD proteins could specifically bind to intracellular CpG DNA in 293T cells.Patients with SLE had a significantly lower methylation level than the controls did(9.32±1.33 vs 11.66±1.04,t=5.68,P<0.05),and the DNA methylation level negatively correlated、with SLE disease activity index(r= -0.78,P<0.01).Condusions There is a lower DNA methylation in patients with SLE than in normal human controls,and the methylation level negatively correlates with SLE disease activity index.     

Analysis of gene probes and gene amplification techniques for diagnosis and monitoring of treatment in childhood leprosy

Nucleic acid sequences of Mycobacterium leprae were detected using gene probes hybridizing with targeting ribosomal RNA (16S rRNA), ribosomal DNA (16S rDNA) and gene amplification techniques (PCR) in skin lesion of paediatric leprosy patients and the effect of treatment on the by these methods. Eighty paediatric leprosy patients were included in the study. Most cases (79%) were between 9 and 16 years of age. Cases were divided into three groups according to treatment status, viz. untreated (30), undergoing treatment (30), and at the end of treatment (20). Clinical and slit smear examination for acid fast bacilli (AFB) was performed and nucleic acids were extracted and fractionated from skin biopsies. M. leprae specific 16S rRNA and 16S rDNA was detected by hybridization with gene probes whereas the 36 kDa gene sequence was detected by a gene amplification assay (PCR). The cases were classified as paucibacillary (PB) and multibacillary (MB) by the standard criteria of WHO (1988). Positivity of 16S rRNA in PB cases decreased from 60% in untreated to 10.5% after 4-8 months of treatment whereas for 16S r DNA, it decreased from 50% to 21%, for PCR from 70% to 36.8% for the same specimen, and all became negative at 1 year. Similar trends were seen in MB cases where positivity in smear positive untreated cases decreased from 100% to 56.2% with 16S rRNA and 42.8% with 16S rDNA and PCR, respectively, after 9-12 months of treatment and all became negative at 2 years, except one case which remained positive with PCR. Similar results were observed in smear negative MB cases, 100% positivity detected by 16S r RNA and PCR, 75% detected by 16S rDNA decreased to zero after 9-12 months of therapy. This study suggests the potential usefulness of gene probes targeting 16S rRNA and 16S rDNA and PCR as supportive molecular tools for diagnosis of smear negative evolving MB disease and also monitoring the response to treatment, these observations however, needs to be validated in prospective follow up studies.     

APPLICATION OF POLYMERASE CHAIN REACTION (PCR) AND PCR BASED RESTRICTION FRAGMENT LENGTH POLYMORPHISM FOR DETECTION AND IDENTIFICATION OF DERMATOPHYTES FROM DERMATOLOGICAL SPECIMENS

Objective:

To develop and optimize polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) targeting 18S rDNA and internal transcribed spacer (ITS) region of fungi for rapid detection and identification of dermatophytes.

Materials and Methods:

Two PCR-RFLP methods targeting 18S rDNA and ITS regions of fungi were optimized using standard and laboratory isolates of dermatophytes and other fungi. Sixty-eight dermatological clinical specimens (nail clippings (56), material obtained from blisters (8), hair root (2), scraping from scaly plaque of foot (1) and skin scraping (1) collected by the dermatologist were subjected to both the optimized PCR-RFLP and conventional mycological (smear and culture) methods.

Results:

PCRs targeting 18S rDNA and the ITS region were sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA, respectively. PCR targeting 18S rDNA was specific for dermatophytes and subsequent RFLP identified them to species level. PCR-RFLP targeting the ITS region differentiated dermatophytes from other fungi with identification to species level. Among the 68 clinical specimens tested, both PCR-RFLP methods revealed the presence of dermatophytes in 27 cases (39.7%), whereas culture revealed the same only in 2 cases (7.40%), increasing the clinical sensitivity by 32.3%. Among 20 smear positive specimens, both PCR-RFLP methods detected dermatophytes in 12 (17.6%). Both the methods detected the presence of dermatophytes in 13 (19.11%) smear and culture negative specimens, increasing the clinical sensitivity by 36.1%.

Conclusion:

PCR-RFLP methods targeting 18S rDNA and the ITS regions of fungi were specific and highly sensitive for detection and speciation of dermatophytes.     

内蒙古地区皮肤化脓性感染部位分离的金黄色葡萄球菌耐药及毒力特征研究

目的:分析皮肤化脓性感染部位分离的金黄色葡萄球菌（简称金葡菌）耐药、毒力及分子流行病学特征,为临床抗感染用药提供实验依据。方法:收集内蒙古医科大学附属医院2020年5-12月皮肤科住院患者皮肤化脓性感染部位及鼻腔分泌物样本进行细菌分离培养。采用基质辅助激光解吸电离飞行时间质谱鉴定疑似金葡菌菌落,用微量肉汤稀释法进行药物...     

Evidence for the presence of bacteria in the blood of psoriasis patients

Evidence exists that microorganisms, particularly in the throat and skin, play a role in the pathogenesis of psoriasis. The aim of this study was to investigate whether evidence for the presence of bacteria, including Streptococcus pyogenes, can be demonstrated in the peripheral blood of patients with guttate and/or chronic plaque psoriasis. Peripheral blood samples from 20 patients with psoriasis, seven guttate, six chronic plaque and seven chronic plaque with associated guttate flare and from 16 control subjects were studied for the presence of bacteria by PCR using universal 16S ribosomal DNA primers and specific primers for S. pyogenes. Sequence analysis of amplified 16S rRNA sequences was used to determine taxonomic identity. Ribosomal bacterial DNA was detected in the blood of all 20 patients with psoriasis, but in none of the controls. Streptococci were detected in six of seven patients with guttate psoriasis, but none had staphylococci. In contrast, staphylococci were identified in 9 of 13 patients with chronic plaque psoriasis, whilst only 2 demonstrated streptococci. In three psoriasis patients, species other than streptococci and staphylococci were identified. These findings suggest that psoriasis is associated with bacteraemia, with distinct taxonomic groups present in guttate and chronic plaque psoriatic subtypes. The causes of the bacteraemia and its implications in psoriasis have yet to be determined.     

PCR直接检测皮肤分枝杆菌感染的研究

目的 探讨PCR直接检测皮肤分枝杆菌感染作为诊断的可能性.方法 PCR检测方法及培养方法(Löwenstein-Jensen培养基)比较分枝杆菌纯菌悬液、模拟皮肤分枝杆菌感染标本前处理前后、疑似皮肤分枝杆菌感染的临床标本前处理前后的检测结果.结果 分枝杆菌纯菌悬液的PCR法和培养法敏感性皆为1×10²个菌细胞/mL.模拟临床标本在经过前处理后在浓度为1×10²个菌细胞/mL的数量级上PCR检测阳性率为60%,而未经过前处理的模拟临床标本在此数量级上PCR检测阳性率为0,培养方法的阳性率为100%,但在浓度为1×10³个菌细胞/mL数量级上PCR检测阳性率为100%.37例临床疑似皮肤分枝杆菌感染标本经前处理后,PCR检测7例阳性,而未经前处理的标本同法检测2例阳性,但培养方法可检出9例阳性.结论 临床皮肤标本经前处理后,PCR检测的敏感性已接近80%,但能明显缩短检测时间.     

Polymerase chain reaction for diagnosis of genital herpes in a genitourinary medicine clinic

BACKGROUND: Polymerase chain reaction (PCR) has well established advantages over culture for diagnosis of herpes viruses, but its technical complexity has limited its widespread application. However, recent methodological advances have rendered PCR more applicable to routine practice. Aim: To compare automated PCR with viral culture for diagnosis of genital herpes. METHODS: We studied 236 patients presenting with clinical features suggestive of genital herpes at an inner city genitourinary medicine clinic. Two swabs were taken from each patient. Cell culture and typing were performed by standard methods. Automated PCR was performed using the LightCycler instrument and the infecting viral type was determined by restriction endonuclease digestion of amplicons. RESULTS: 109 patients (46%) had a positive test for herpes simplex virus (HSV). In 88, both PCR and culture were positive; in 21 PCR only was positive. With both detection methods, lesion duration and morphology were associated with HSV detection. Compared with culture alone, use of PCR increased sensitivity by 13.3% in specimens from vesicular lesions, by 27.4% from ulcerative lesions, and by 20.0% from crusting lesions. CONCLUSIONS: We advocate adoption of automated PCR as an efficient HSV detection and typing method for diagnosis of genital herpes in routine clinical practice. PCR allowed rapid laboratory confirmation of the diagnosis and increased the overall HSV detection rate by 24%.     

Detection of Mycobacterium tuberculosis complex DNA by the polymerase chain reaction for rapid diagnosis of cutaneous tuberculosis

Summary We assessed the polymerase chain reaction (PCR) technique to detect Mycobacterium tuberculosis complex DNA In 48 paraffin-embedded specimens from 32 patients with different variants of cuttineous tuberculosis, and compared the resuts with those of culture. A 123 bp product of the 1S6110 insertion sequence specific of M. tuberculosis complex was amplified and confirmed by digestion with Sall restriction endonuclease. The time required for the procedure was 3 days. Thirty-seven samples (77.1%) were positive for M. tuberculosis complex DNA. No false positive results were obtained in nine negative controls, Of the 20 specimens tested by PCR and culture, the frequency of positivity was 90% for DNA amplification and 65% for culture. In seven cases of lupus vulgaris, the figures were 100% and 57% respectivety. In the 11 specimens culture negative or not microbiologically tesled and PCR negative, evidence for tuberculous infection was provided hy the correlation of various relative fmd absolute criteria. These results show that PCR amplification of the IS6110 insertion fragment is a rapid and accurate means for the detection of M. tuberculosis complex DNA in paraffin-embedded skin biopsies from patients with cutaneous tuberculosis, especially in paucibacillary lesions.