应用DNA微阵列技术快速鉴定分枝杆菌菌种

目的利用DNA微阵列的高通量、高效性,建立一种快速、简便的分枝杆菌分子菌种鉴定方法,为临床医师正确诊断提供依据。方法以DNA直接测序法为对照,通过PCR-SSCP和DNA微阵列技术分析28种分枝杆菌标准菌株、9种非分枝杆菌和465株分枝杆菌临床分离株的菌种。结果应用DNA微阵列技术分析28种分枝杆菌标准菌株和9种非分枝杆菌菌株,特异性100％。465株分枝杆菌临床分离株中,经16S rRNA PCR-SSCP初步菌种鉴定,256株为结核分枝杆菌复合群,应用DNA微阵列分析,显示与分枝杆菌属探针M和结核分枝杆菌复合群探针a杂交阳性,两种鉴定方法结果一致;209株PCR-SSCP初步鉴定为非结核分枝杆菌的分离株,经芯片分析,68株为龟分枝杆菌龟亚种和脓肿亚种,46株为胞内分枝杆菌,34株为堪萨斯、瘰疬、胃和猿猴分枝杆菌复合群,31株为偶然分枝杆菌,16株为戈登分枝杆菌,3株为鸟分枝杆菌,2株为海和溃疡分枝杆菌复合群,1株为土分枝杆菌, 1株为迪氏分枝杆菌,1株为草分枝杆菌;另6株只与探针M杂交,经测序显示5株为胞内分枝杆菌,但其基因序列与标准菌株不完全相同,1株为新金色分枝杆菌,芯片上无鉴定该菌种的探针。结论用DNA微阵列可简便、快速、灵敏、特异地将大多数分枝杆菌鉴定到种,提高分枝杆菌病的正确诊断率,指导临床合理治疗。     

应用反向斑点杂交技术快速鉴定脊柱结核分枝杆菌菌种

目的探讨反向斑点杂交技术在脊柱结核分枝杆菌菌种鉴定中的应用价值。方法通过16SrDNA聚合酶链反应一单链构象多态性（polymerasechainreaction—singlestrandconformationpolymorphism,PCR—SSCP）分析鉴定37例脊柱结核术后患者病理组织或脓液抗酸染色阳性标本的分枝杆菌菌种;设计与合成用于鉴定分枝杆菌菌种的16SrDNA寡核苷酸探针,制作硝酸纤维膜微阵列,与待鉴定菌株生物素标记的16SrDNA基因PCR产物进行反向斑点杂交。结果37例脊柱结核标本中,经16SrDNAPCR—SSCP初步鉴定,36株为结核分枝杆菌复合群,1株为非结核分枝杆菌;经杂交分析,34株结核分枝杆菌分离株鉴定为结核分枝杆菌复合群,1株鉴定为偶发分枝杆菌,分别与相应的特异探针杂交,2株与分析探针杂交阴性。结论16SrDNAPCR一反向斑点杂交技术可用于陕速鉴定脊柱结核分枝杆菌菌种。     

2017年绍兴地区106株非结核分枝杆菌菌种鉴定结果分析

目的 分析绍兴地区非结核分枝杆菌(NTM)的流行情况,为NTM诊疗提供科学依据。方法 对绍兴地区2017年临床分离的972株分枝杆菌,采用结核分枝杆菌抗原检测胶体金法进行结核分枝杆菌复合群和NTM的初步菌种鉴定,结果106株为NTM,占分枝杆菌培养阳性菌株的10.91%(106/972),对初步鉴定为NTM的菌株应用 DNA微阵列芯片法进行NTM菌种鉴定。结果 106株NTM菌株经DNA微阵列芯片法鉴定,共有14个菌种,其中菌株数量前3位的菌种为:胞内分枝杆菌[55株(51.89%)]、鸟分枝杆菌[19株(17.92%)]、堪萨斯分枝杆菌[13株(12.26%)]。其余分别为:龟-脓肿分枝杆菌[4株(3.77%)]、瘰疬分枝杆菌[3株(2.83%)]、戈登分枝杆菌[3株(2.83%)]、土分枝杆菌[2株(1.89%)]、其他[7株(6.60%)]。结论 2017 年绍兴地区NTM 临床分离率占分枝杆菌培养阳性菌株的10.91%;NTM种类多,以胞内分枝杆菌、鸟分枝杆菌、堪萨斯分枝杆菌为主要流行菌种。     

福建省临床分离非结核分枝杆菌菌种分布研究

目的 分析我院临床分离非结核分枝杆菌(NTM)菌株,探讨福建省NTM临床分离率和菌种分布情况。方法 对我院2009-2012年临床分离6 362株分枝杆菌进行结核分枝杆菌复合群(MTBC)与NTM鉴别,对鉴定为NTM菌株应用hsp65-和rpoB- PCR-RFLP进行菌种鉴定。结果 6 362株临床分离分枝杆菌共鉴别出649株NTM。鉴别出的649株NTM菌株,经hsp65-和rpoB-PCR-RFLP鉴定,菌种分布达24个种,其中菌株数量前3位菌种为:胞内分枝杆菌(M.intracellulare)302株(46.5%),鸟分枝杆菌(M. avium)138株(21.3%),脓肿分枝杆菌(M. abscessus)81株(12.5%)。结论 2009-2012 年福建省NTM临床分离率占分枝杆菌培养阳性菌株的10.2%,NTM种类多,以鸟-胞内分枝杆菌复合群(MAC)为福建省NTM的主要流行菌种,占总数的67.8%。     

129株非结核分枝杆菌采用两种分子检测技术行菌种鉴定的结果分析

目的 分析北京地区3个单位非结核分枝杆菌 (NTM)临床分离株采用芯片杂交法(简称“芯片法”)和16S rRNA基因测序(简称“基因测序”)进行菌种鉴定的结果,为临床分枝杆菌病的快速准确诊断提供科学依据。方法 用芯片法和基因测序进行北京地区3个单位129株NTM临床分离株的菌种鉴定,实验操作者之间采用双盲法,对两种鉴定方法的结果进行比较和评价。结果 129株NTM菌株中,有107株进行分枝杆菌基因测序,其中59株为胞内分枝杆菌(55.1%);29株为堪萨斯分枝杆菌(27.1%);5株为龟分枝杆菌/脓肿分枝杆菌(4.7%);3株为鸟分枝杆菌(2.8%);2株为偶发分枝杆菌(1.9%);2株为戈登分枝杆菌(1.9%);1株为蟾蜍分枝杆菌(0.9%);1株为草分枝杆菌(0.9%);1株为瘰疬分枝杆菌(0.9%);4株为其他NTM(3.7%)。有116株进行芯片法分枝杆菌菌种鉴定,且有96株同时进行芯片法和基因测序法检测,两种方法进行菌种鉴定的符合率为88.5%(85/96)。基因测序发现有3株外来菌株,分别为明尼苏达分枝杆菌、熊本分枝杆菌、提门分枝杆菌(M.temen)。结论 北京3家机构NTM菌种以胞内分枝杆菌、堪萨斯分枝杆菌和龟分枝杆菌/脓肿分枝杆菌为主。用芯片法和基因测序对NTM临床分离株进行菌种鉴定的一致率较高。     

PCR测序和基因芯片快速鉴定分枝杆菌菌种的应用研究

目的 评价2种分枝杆菌菌种快速鉴定方法的临床应用价值。方法 分别用16S～23S rDNA转录间隔序列（ITS）PCR-直接测序和基于16S rRNA基因的基因芯片检测21株分枝杆菌标准菌株和50株临床分离株。结果 21株分枝杆菌标准株中,ITS PCR测序法将18株准确鉴定到种,结核分枝杆菌、非洲分枝杆菌菌株只能鉴定到结核分枝杆菌复合群,海分枝杆菌无法鉴别是海分枝杆菌还是溃疡分枝杆菌;芯片法正确鉴定16株,1株胃分枝杆菌鉴定为堪萨斯分枝杆菌,3株不在芯片检测范围内。50株临床菌株中, 47株2种方法结果一致,1株不在芯片检测范围内。结论 基因芯片鉴定分枝杆菌快速、特异,准确性高,临床常规开展应用需要进一步研究。     

山东省非结核分枝杆菌感染的菌种分布研究

目的研究山东省13个哨点县2004—2007年非结核分枝杆菌临床分离率和菌种分布情况。方法对山东省13个哨点县2004—2007年送到汉光中心的2625株分枝杆菌菌株,进行分枝杆菌菌群鉴定试验,鉴定为非结核分枝杆菌的菌株应用16SrDNA测序方法进行菌种鉴定。结果2625株菌株分枝杆菌菌群鉴定39株为非结核分枝杆菌菌群,39株菌株16SrDNA序列分析结果有36株是非结核分枝杆菌,其中29株为胞内分枝杆菌株（80.6%）, 其余分别为堪萨斯分枝杆菌、偶然分枝杆菌各2株、戈登分枝杆菌、龟脓肿分枝杆菌复合物、瘰疬分枝杆菌各1株;结核分枝杆菌复合群2株;鼻疽诺卡氏菌1株。山东地区非结核分枝杆菌临床分离率占分枝杆菌培养阳性菌株的1.4%。结论山东地区流行的非结核分枝杆菌以慢生长分枝杆菌的胞内分枝杆菌为主。     

hsp65 and rpoB PCR-RFLP用于龟/脓肿分枝杆菌复合群种的快速鉴定(英文)

目的探讨和评价hsp65and rpoB PCR-RFLP用于龟/脓肿分枝杆菌复合群种的快速鉴定。方法收集经PNB/TCH鉴别培养基表型鉴定和16s rRNA基因测序鉴定为龟/脓肿分枝杆菌复合群的临床分离菌株,用hsp65and rpoBPCR-RFLP进行种/亚种鉴定。结果经表型鉴定为非结核分枝杆菌的27株临床菌株,16s rRNA基因测序分析与龟/脓肿分枝杆菌的同源性达到99.7%。经hsp65PCR-RFLP and rpoBPCR-RFLP鉴定18株为脓肿分枝杆菌(M.abscessus),4株为溃疡分枝杆菌(M.absecces),另5株表现为独特的指纹特征,可能是一个新的亚种。结论能够快速进行龟/脓肿分枝杆菌复合群种/亚种的鉴定。     

分枝杆菌临床分离株菌种的分子鉴定

目的 建立对临床分离分枝杆菌菌种快速、精确鉴定的方法学平台,了解分枝杆菌菌种的分布,为结核分枝杆菌(MTB)和非结核分枝杆菌(NTM)病临床精确诊断和有效治疗提供依据.方法 330株分枝杆菌分离株来自于2007年5月至2008年12月在哈尔滨市胸科医院诊断为结核病的住院患者.菌株灭活后提取基因组DNA,采用PCR方法,结合PCR-限制性片段长度多态性(RFLP)和DNA序列测定方法进行菌种精确鉴定.结果 330株临床分离菌株全部精确鉴定,其中328株[占99.4％(328/330)]为结核分枝杆菌复合群(MTBC),2株[占0.6％(2/330)]为NTM.328株MTBC中,MTB 326株、非洲分枝杆菌(M.African)1株、田鼠分枝杆菌(M.microti)1株,分别占MTBC的99.4％ (326/328)、0.3％(1/328)和0.3％(1/328).经测序和基因组同源性分析发现,2株NTM为胞内分枝杆菌(MAC),与MAC的同源性分别为99％和93％,2株菌之间的同源性为93％.结论 建立了临床分离分枝杆菌菌种快速鉴定的方法学平台,临床诊断为结核病的患者MTB比例占绝对优势,也发现有M.African、M.microti和NTM感染.     

DNA芯片鉴定分枝杆菌的研究

目的评价DNA芯片技术鉴定分枝杆菌菌种的应用价值,为临床实际应用提供科学依据。方法收集以BACTECMGIT960从结核病患者分离到的分枝杆菌阳性培养物,以传统分枝杆菌菌种鉴定方法为对照,应用DNA芯片技术进行菌种鉴定。结果两种方法鉴定结果一致和基本一致共112株,吻合率为83.6%(112/134),包括胞内分枝杆菌50株,龟分枝杆菌14株,结核分枝杆菌14株,戈登分枝杆菌9株,鸟分枝杆菌7株,偶然分枝杆菌7株,堪、瘰、胃和猿分枝杆菌6株,土分枝杆菌和海分枝杆菌各1株;未分类NTM3株。应用DNA芯片未能分类的17株分枝杆菌中,传统方法分别鉴定为戈登分枝杆菌5株、胞内分枝杆菌3株、龟分枝杆菌2株、蟾分枝杆菌、耻垢分枝杆菌和浅黄分枝杆菌各1株,未分类NTM4株。结论用DNA芯片检测技术,可以简便、快速、灵敏、特异地将大多数分枝杆菌鉴定到种,但有待进一步完善。     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.