聚合酶链反应和免疫组化检测猫抓病患者淋巴结巴尔通体感染

目的探讨聚合酶链反应(polymerase chain reaction,PCR)和免疫组化法检测猫抓病患者巴尔通体感染情况,探讨两种方法在石蜡包埋组织中确诊猫抓病的实用价值。方法收集94例经病理形态学诊断的猫抓病石蜡包埋淋巴结组织,分别使用针对巴尔通体柠檬酸合成酶(glt A)基因、16~23S rRNA基因的2种引物扩增巴尔通体基因序列以及使用汉赛巴尔通体单克隆抗体检测组织中巴尔通体感染情况。结果 66例(70.2%)抗汉赛巴尔通体单克隆抗体染色阳性,阳性信号主要呈点状、颗粒状,少数呈线样勾勒出细菌形状。应用PCR法检测有57例(60.6%)汉赛巴尔通体191 bp长度的glt A基因;另40例(42.5%)检测出汉赛巴尔通体163 bp长度的16~23S rRNA基因,无其他巴尔通体种类检测出。综合上述检测结果显示有76例(80.8%)检出汉赛巴尔通体的感染。结论汉赛巴尔通体是我国猫抓病患者的病原体,应用PCR法和免疫组化En Vision两步法检测汉赛巴尔通体有助于确诊猫抓病,汉赛巴尔通体单克隆抗体检测是目前较为理想的检测方法。     

猫抓病性淋巴结炎的临床病理分析

目的：探讨猫抓病性淋巴结炎的诊断和鉴别诊断要点,方法：应用免疫组化S－P法对26例猫抓病性淋巴结炎分别标记CD20、CD3、CD68;15例进行Warthin-Starry银染色,结果：早期微脓生肉芽肿21例,其病变特征是微脓肿和肉芽肿形成。淋巴结早期微脓肿周围为CD20＋的B淋巴细胞和CD68＋组织细胞,当典型微脓肿性肉芽肿形成时,CD68＋的类皮细胞呈放射状排列,其间夹有CD3＋T淋巴细胞;15例中12例Warthin-Starry染色（＋）。结论：猫抓病灶性淋巴结炎的主要特征是微脓肿性肉芽肿形成,免疫组化标记有助于识别病变中反应增生的细胞成分,本病可能与细菌感染所诱导的细胞免疫反应有关。     

Warthin-Starry染色的应用及体会

虽然目前分子技术在临床病理诊断领域中取得了广泛应用,但许多传统的特殊染色法仍是检测病原体不可缺少的方法.其中,Warthin-Starry(W-S法)染色就是一种经典的特染方法.W-S法首先在1920年由Warthin和Starry[1]作为显示螺旋体的一种特殊染色方法提出,其原理是一种嗜银反应.下面我们将结合相关文献及自身体会就该方法的主要环节及综合应用作简要介绍.     

中哈边境阿拉山口口岸小型兽类感染巴尔通体调查研究

为研究阿拉山口口岸小型兽类巴尔通体感染情况,对捕获的75只小型兽类进行形态学鉴定后,提取肝脏和脾脏核酸扩增巴尔通体Bartonella枸橼酸合酶基因(gltA),通过阳性序列的比对进行巴尔通体分子鉴定和序列分析。结果显示,阿拉山口口岸小型兽类巴尔通体感染率为9.33%(7/75),分别为大沙鼠3只、小家鼠2只、红尾沙鼠1只、小林姬鼠1只。经测序,7份阳性样本获得的巴尔通体分属于伊丽莎白巴尔通体、格拉汉姆巴尔通体、抚远巴尔通体和泰勒巴尔通体4种,其中3个阳性为伊丽莎白巴尔通体,各鼠种巴尔通体感染率无显著性差异。伊丽莎白巴尔通体和格拉汉姆巴尔通体为鼠传人类致病性巴尔通体,应通过灭鼠、灭蚤以降低巴尔通体自然宿主或传播媒介的密度,防止巴尔通体在国境口岸的传入传出。     

内蒙古口岸蜱类及其自然感染病原体监测

2012～ 2014年在内蒙古4个口岸采用人工布旗法监测游离蜱,并对其自然感染的病原体进行检测.共获得3 502只蜱,随机抽检其中的836只,共检出9种病原微生物,包括立克次体196只(44.2％)、嗜吞噬细胞无形体73只(16.5％)、新疆出血热病毒57只(12.9％)、贝纳柯克斯体43只(9.7％)、土拉菌33 (7.4)、伯氏疏螺旋体20只(4.5％),另外还有巴尔通体10只、森林脑炎病毒9只和新布尼亚病毒2只.此外,抽检蜱种首次在内蒙古口岸发现巴尔通体自然感染,因而,巴尔通体病的监测和控制值得关注,并持续开展多种蜱传疾病的防治工作.     

华南猫抓病2例

对猫抓病的研究已有70余年,以前疑是由病毒引起,1983年美国病理学家Wear用Warthin—Starry银染色发现其病原为一种嗜银染色的杆菌。猫抓病的主要临床和病理表现为：有猫抓伤史,皮肤丘疹,低热和局部淋巴结肿痛,可自愈,也可进展为重症或复发型;组织学上淋巴结炎呈阶段病变表现：开始为窦性组织细胞增生,以后形成非坏死性结核样结节,结节可增大、融合并且中央组织坏死,白细胞集聚形成星形脓肿（stellme abscess）,其外层为结核样肉芽肿。     

云南省贡山地区小型兽类几种细菌性病原体携带现况调查

为掌握云南贡山地区野生小型兽类的种类和主要细菌性病原体的携带情况,于2014—2015年在该地区诱捕野生小型兽类,进行种类鉴定,解剖取肺、脾等组织。采用巢式PCR对无形体Anaplasmataceae、恙虫病东方体Orientia tsutsugamushi、斑点热群立克次体(Spotted fever group rickettsiae,SFGR)以及莱姆病螺旋体Lyme disease spirochete等4类病原体进行检测。结果共捕获小兽759只,涉及14属35种,优势属为姬鼠属(29.1%)和绒鼠属(17.4%)。对其中541份标本进行无形体PCR检测,结果显示:12份新埃立克体(Candidatus Neoehrlichia mikurensis)阳性,来自姬鼠、绒鼠、鼩鼱和白腹鼠;2份沃尔巴克氏体Wolbachia阳性,来源于鼩鼱和灰腹鼠;3份巴尔通体Bartonella阳性,来源于绒鼠和大足鼠。采用56 k Da基因片段引物对恙虫病东方体进行检测,发现4份阳性标本。其中2份与Boryong血清型菌株同源性最高,均为97.0%;1份与Karp血清型同源性最高,为86.0%;另一份与Kato-related血清型同源性最高,为86.0%,系统发生树显示,其与Kato-related位于同一分支。提示这两份标本可能是恙虫病东方体Karp血清型和Kato-related血清型菌株的变异株。     

咽部特殊感染症病理特点及病原体检测的探讨

目的 探讨咽部特殊感染性疾病的病理特点,研究其病原体的检测方法,以提高特殊感染症的诊断水平.方法 分析北京同仁医院1998年1月至2008年1月咽部61例黏膜溃疡病患者的临床病理资料,以简易的组织芯片染色技术,对61例咽部黏膜溃疡组织,用PAS、Giemsa、革兰、美蓝、改良的Warthin-Starry(W-S)及抗酸染色进行集中染色,比较各种染色方法对病原体的染色效果,找出不同病原体的最佳染色方法.结果 组织切片内发现密螺旋体23例,短螺旋体10例,分枝杆菌4例,鼻硬结杆菌4例,霉菌侵袭1例,细菌和白色念珠菌混合感染1例,扁桃陷窝放线菌2例.非特异性细菌感染16例.结论 螺旋体、鼻硬结杆菌均以改良的W-S染色效果最佳,真菌染色应根据真菌的不同类型联合应用革兰及PAS、W-S染色进行鉴别,分枝杆菌以传统的抗酸染色效果最好.     

3种特殊染色在肺慢性肉芽肿性炎中的应用

正肺部肉芽肿性炎症通常由于肺部感染了特殊的病原微生物或寄生虫形成有相对诊断意义的特征性肉芽肿。常见的病原体有结核杆菌、伤寒杆菌、梅毒螺旋体、真菌等。本文采用过碘酸-Schiff(PAS)染色法、Grocott六胺银染色法、Ziehl-Neelesn抗酸杆菌染色法,并改良其染色技术,以帮助鉴别这些病原体感染导致的肺部疾病。1材料与方法1.1材料收集上海市胸科医院存档的肺慢性肉芽肿性炎     

抗人骨肉瘤单克隆抗体5D3的特异性鉴定

目的 鉴定抗人骨肉瘤单克隆抗体（mAb)5D3的特异性。方法 用免疫组化染色法检测mAb 5D3与骨肉瘤细胞系、骨肉瘤组织、其它肿瘤组织及正常组织的反应性。结果 免疫组化染色检测发现，mAb 5D3相应抗原在部分骨源性肿瘤，特别是骨肉瘤组织及骨肉瘤细胞系呈高度表达，中度以上染色者占阳性总数的77％以上，主要分布于细胞核上。14种正常组织中未见阳性反应。结论 mAb 5D3具有高度特异性，为骨肉瘤的早期诊断及免疫治疗提供了可能性。     

Cat scratch disease

Summary The aetiological agent of cat scratch disease (CSD) has been unknown for more than 30 years. Recently, a micro-organism clearly shown with Warthin-Starry silver (W-S) stain was found and thought to be a possible cause of the disease. In this study, 32 cases of regional lymphadenopathy histologically compatible with CSD and 20 contrasting cases of lymphadenopathy were examined retrospectively with W-S stain. W-S positive pleomorphic organisms were clearly demonstrated in 20 of the 32 suspected cases of CSD, but in none of the other cases. The onset of disease in these 20 cases with W-S positive organisms occurred between July and January. This seasonal variation in the onset of disease was highly significant (P<0.005) and was not due to a single epidemic. Moreover, some characteristic morphological features of the organism were found by electron microscopic observations. Ultrastructurally, the organism was a bacterium showing a chain-like arrangement, septal formation, branching and clubbed ends.     

Isolation of Bartonella henselae DNA from the peripheral blood of a patient with cat scratch disease up to 4 months after the cat scratch injury

We report the case of a girl with cervical lymphadenitis and a persistent primary lesion of cat scratch disease (CSD). Bartonella henselae DNA was isolated from plasma samples collected 3 and 4 months after the cat scratch, indicating that recurrent and long-term shedding of Bartonella DNA into peripheral blood may occur in typical CSD.     

Intracellular location of Bartonella henselae cocultivated with Vero cells and used for an indirect fluorescent-antibody test.

Bartonella henselae, the major causative agent of cat scratch disease, was cocultivated with Vero cells on chamber slides and visualized by indirect immunofluorescence by using a patient serum containing specific antibodies. Confocal microscopy localized the granular B. henselae-specific fluorescence mainly around the nuclei of Vero cells. By transmission electron microscopy, these granules were identified as clusters of multiple intracellular organisms. Fixed slides with the monolayers of Vero cells with intracellular B. henselae were used for an indirect fluorescent-antibody test to investigate the seroprevalence of specific immunoglobulin G in 100 serum samples from blood donors. Seventy-four serum samples were negative; 19, 3, and 4 were positive at dilutions of 1:64, 1:128, and 1:256, respectively. In our population, a serum titer of 1:256 or greater should stimulate further investigations. Moreover, elucidation of the mechanism by which B. henselae enters the cells may help to understand the pathogenesis of cat scratch disease.     

Ultrastructure of Warthin-Starry stain-positive bacteria in abscess-forming reticular lymphadenitis.

This study describes the ultrastructure of Warthin-Starry (WS) stain-positive bacteria in abscess-forming reticular lymphadenitis (ARL) compatible with cat scratch disease (CSD). Sections containing WS-positive bacteria were re-embedded in Epon, and semithin sections were examined by electron microscopy. Silver particles were aggregated on the outer surfaces of the bacteria. Stereoscopic observations clearly showed that the bacteria were pleomorphic, rod-shaped, arranged in a row or at an angle, and frequently showed septal formation. Electron microscopy of ultrathin sections revealed that the cell wall possessed an outer membrane characteristic of gram-negative bacteria. The results indicate that rod-shaped bacteria with the WS-positive, gram-negative staining cause ARL histopathologically consistent with CSD.     

Evaluation of indirect fluorescence antibody assay for detection of Bartonella clarridgeiae and Seroprevalence of B. clarridgeiae among patients with suspected cat scratch disease

The possibility of Bartonella clarridgeiae being a causative agent of cat scratch disease (CSD) was investigated by using indirect fluorescence antibody assays with 288 suspected CSD patients. Immunoglobulin G antibody to noncocultivated B. clarridgeiae was suitable only for detection of B. clarridgeiae antibody. Significant cross-reactivity between Bartonella henselae and B. clarridgeiae was noted, and no CSD case caused by B. clarridgeiae was detected.     

Cat-scratch disease: epidemiology, aetiology and treatment.

Cat-scratch disease (CSD) is a clinical syndrome that usually presents as a self-limiting lymphadenopathy associated with a cat scratch or bite. Commonly affecting children and young adults, it has a worldwide distribution. In temperate climates, higher rates are reported in the autumn and winter, which can be attributed to the seasonal breeding of the domestic cat. The organism responsible was identified in 1983, having eluded detection for 50 years. Initially, Afipia felis was thought to be the cause; however, subsequent study failed to confirm a link. During the 1990s, it was demonstrated conclusively that Rochalimaea henselae, later reclassified as Bartonella henselae, was the cause of CSD. B. henselae has been isolated from bacteraemic cats, with transmission among cats thought to be via the cat flea. Although other Bartonella species are transmitted by arthropod vectors, it is unlikely that the cat flea is involved directly in human infection, but plays a role in amplifying the reservoir. B. henselae is difficult to culture, and either serology or the polymerase chain reaction are considered to be the best methods of detection. Genetic variation occurs amongst B. henselae strains, perhaps explaining the inconsistency of some diagnostic techniques. A separate serogroup (Marseilles) has been reported in a seronegative patient with CSD, and B. clarridgeiae has the potential to cause the disease. Atypical presentation is seen in up to 25% of cases, and manifests itself as ocular involvement, encephalopathy, granulomatous hepatitis, hepatosplenic infection, endocarditis and osteomyelitis. The majority of CSD cases resolve spontaneously and do not require antibiotic treatment. In complicated CSD, treatment with trimethoprim-sulphamethoxazole, ciprofloxacin or azithromycin is recommended, with gentamicin being reserved for the severely ill patient.     

Diagnosis of cat scratch disease with Bartonella henselae infection in formalin-fixed paraffin-embedded tissues by two different PCR assays.

ABSTRACT: Cat scratch disease (CSD) is commonly caused by Bartonella henselae infection. Clinical history and histologic findings are often insufficient to establish a definitive diagnosis of CSD. We retrospectively studied formalin-fixed, paraffin-embedded (FFPE) lymph nodes from 35 patients with histologically suspected CSD by 2 different PCR assays and immunohistochemistry (IHC). The first primer pair amplified a 163-bp fragment of the 16S rRNA gene in 19 of the 35 cases (54%). The second primer pair amplified a 191-bp fragment of the henselae citrate synthase (gltA) gene in 17 of the 35 cases (49%). IHC identified the organisms in 8 of 33 cases (24%). Fresh cultures of various Bartonella species showed a specific PCR product with an analytical sensitivity of 0.5 to 5 pg bacterial DNA. Bartonella species were identified by the unique size of the amplified PCR product. Twenty-two lymph nodes without morphologic evidence or a history of CSD were negative by PCR and immunostaining. Tissues from a patient with Legionella pneumophila were also negative by PCR and immunostaining for CSD supporting the specificity of the PCR reaction. The specific PCR products of the B. henselae were confirmed by sequencing. Human beta-actin for each case was amplified to check the integrity of the DNA. Our data indicate that detection of Bartonella DNA by PCR is useful to confirm the diagnosis of CSD.     

Combining cytomorphology and serology for the diagnosis of cat scratch disease

Cat scratch disease (CSD) is a self limited zoonotic disease that presents most commonly as a regional lymphadenopathy. We are reporting a case of a 25-year-old male patient who presented with fever and large right inguinal lymphadenopathy. The diagnosis of cat scratch disease was confirmed based on the characteristic cytopathological features on aspirate smears from the lymph node and the serological titers for Bartonella henselae. This case report emphasizes the importance of combining Bartonella serology, and cytopathology in the diagnostic work-up of febrile lymphadenopathy and suspected CSD since the culture of this organism is arduous.     

Genomic variation of Bartonella henselae strains detected in lymph nodes of patients with cat scratch disease

Bartonella henselae is the primary agent of cat scratch disease (CSD). In order to study the genetic variation of B. henselae and the correlation of the various genotypes with epidemiological and clinical findings, two seminested, groEL- and pap31-based PCR assays were carried out with specimens from 273 patients. Amplicons were sequenced to determine the genotype of the causative Bartonella species. Compared to our reference intergenic spacer region-based PCR, the groEL- and pap31-based assays were 1.7 and 1.9 times more sensitive, respectively. All 107 positive patients were infected with B. henselae; neither Bartonella clarridgeiae nor other species were detected. Based on the groEL and pap31 sequences, B. henselae amplicons were classified into two genogroups, Marseille and Houston-1, and into four variants, Marseille, CAL-1, Houston-1, and a new variant, ZF-1. Patients infected with either one or the other genogroup did not exhibit different epidemiological or clinical characteristics. Our study highlights the genotypic heterogeneity of B. henselae in patients with CSD.     

Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR.

A PCR assay was developed by using degenerate primers that allow amplification of a 414-bp fragment of DNA from the rickettsia-like organisms Rochalimaea henselae and R. quintana. Internal oligonucleotides were used as hybridization probes, permitting rapid differentiation of these two Rochalimaea species. DNAs from 12 different isolates of R. henselae were amplified with the PCR primers, and the resulting 414-bp PCR product hybridized only with the R. henselae-specific probe. DNAs from four different isolates of R. quintana were amplified and produced a PCR product of the same size that hybridized only with the R. quintana-specific probe. DNAs from isolates of R. elizabethae, R. vinsonii, Bartonella bacilliformis, and Afipia felis failed to amplify the 414-bp fragment in the PCR assay. This two-step assay was applied to DNAs extracted from 16 fresh (unfixed) lymph node biopsy specimens and nine aspirates from patients with clinical cat scratch disease (CSD) to assay for the presence of R. henselae or R. quintana DNA in these samples. Twenty-one (84%) of 25 lymph node samples from CSD patients were positive for R. henselae, while none were positive for R. quintana. The characteristic 414-bp fragment was not amplified from eight lymph node tissue samples from non-CSD cases. These results provide evidence that R. henselae, and not R. quintana, plays the central role in the etiology of CSD.