超声引导下细针抽吸细胞学检查联合BRAF V600E基因检测诊断甲状腺影像报告和数据系统(TI-RADS)4类结节

目的 观察超声引导下细针抽吸细胞学检查（FNAC）联合BRAF V600E基因检测对甲状腺影像报告和数据系统（TI-RADS）4类结节的诊断价值。方法 回顾性分析152例TI-RADS 4类结节的超声、FNAC、术后病理及BRAF V600E基因检测等资料。FNAC以Bethesda报告系统分类≥Ⅴ类为标准诊断甲状腺癌。检测BRAF突变,以阳性预测甲状腺癌。以病理结果为金标准,分别计算FNAC、BRAF V600E基因检测及FNAC联合BRAF V600E基因检测诊断甲状腺癌的敏感度、特异度和准确率。结果 BRAF V600E基因检测及超声引导下FNAC联合BRAF V600E基因检测诊断甲状腺癌的敏感度、特异度、准确率均明显高于单纯FNAC （P均<0.05）。FNAC对26个结节（26/152,17.11%）难以明确其良恶性,联合BRAF V600E基因检测后均获明确诊断,诊断敏感度、特异度及准确率分别为95.45%、100.00%及96.15%。结论 超声引导下FNAC联合BRAF V600E基因检测预测TI-RADS 4类结节良恶性优于FNAC,尤其对于FNAC难以明确性质的结节有较高临床价值。     

甲状腺乳头状癌BRAF V600E基因突变与超声及临床特征的相关性研究

目的:探讨甲状腺乳头状癌(PTC)BRAF V600E基因突变与超声及临床特征的相关性。方法:选取2020年6月—2022年7月湖南省人民医院手术病理证实为PTC患者627例,共737个结节,分为BRAF V600E(+)组和BRAF V600E(-)组,比较两组的基线资料。采用单因素及多因素Logistic回归分析与PTC BRAF V600E基因突变相关的超声及临床特征。结果:BRAF V600E基因突变率为71.8%。BRAF V600E(+)组和BRAF V600E(-)组在中央区淋巴结转移、颈侧区淋巴结转移、包膜外浸润、超声最大径、病理直径及PTC结节的回声、形态、边缘、微钙化方面的差异有统计学意义(P <0.05)。单因素及多因素Logistic回归分析显示,颈侧区淋巴结转移、包膜外浸润、PTC结节形态及微钙化是BRAF V600E基因突变的独立危险因素。结论:颈侧区淋巴结转移、包膜外浸润、甲状腺结节形态及微钙化与PTC患者BRAF V600E基因突变相关。对PTC的超声及临床特征进行评估可为患者治疗方案的制定提供帮助。     

甲状腺乳头状癌超声图像特征及BRAF V600E突变与颈部淋巴结转移的关系

目的 分析甲状腺乳头状癌（PTC）超声图像特征及BRAF V600E突变与颈部淋巴结转移的关系。方法 选取于我院行甲状腺癌根治术患者316例,根据甲状腺癌术后病理分期（pTNM）结果,将pN0（无颈部淋巴结转移）者归为未转移组（160例）,pN1（有颈部淋巴结转移）者归为转移组（156例）,比较两组超声图像特征、临床资料、BRAF V600E突变情况的差异。采用多因素Logistic回归分析PTC超声图像特征、BRAF V600E突变及各临床资料与颈部淋巴结转移的关系。结果 转移组98例（62.8%）发生BRAF V600E突变,未转移组121例（75.6%）发生BRAF V600E突变,两组BRAF V600E突变占比比较,差异有统计学意义（P<0.05）。转移组与未转移组年龄及病灶数目、大小、沙粒样钙化、纵横比、边缘、与被膜关系、累及腺叶比较,差异均有统计学意义（均P<0.05）。多因素Logistic回归分析显示,病灶大小、纵横比、与被膜关系、累及腺叶均为颈部淋巴结转移的危险因素（OR=3.606、4.061、2.149、8.578,均P<0.05）,年龄、沙粒...     

甲状腺乳头状癌中BRAF~(V600E)基因突变与超声声像图的相关性研究

目的探讨甲状腺乳头状癌中BRAF~(V600E)基因与甲状腺乳头状癌声像图特征的关系。方法采用ARMS法检测91例共117个病灶的甲状腺乳头状癌(PTC)患者手术切除的甲状腺及其癌旁组织BRAF~(V600E)基因突变情况,分为BRAF~(V600E)基因突变组与野生组,比较两组超声声像图特征。结果 88个(75.2%)PTC癌组织标本发生BRAF~(V600E)基因突变,29个(24.8%)未发生突变。PTC BRAF~(V600E)基因突变组与野生组两组甲状腺声像图结节的边缘和颈部异常淋巴结差异有统计学意义(P0.05)。结论 PTC中BRAF~(V600E)基因突变率较高;BRAF~(V600E)基因突变与PTC超声声像图特征有相关,其中结节边缘不光整和有颈部异常淋巴结特征的组织中BRAF~(V600E)基因突变发生率高。     

甲状腺乳头状癌BRAF V600E突变及超声特征与颈部淋巴结转移的相关性研究

目的探讨甲状腺乳头状癌(papillary thyroid carcinoma, PTC)患者鼠类肉瘤滤过性毒菌致癌同源体B(v-Raf murine sarcoma viral oncogene homolog B, BRAF) V600E基因突变及超声特征与颈部淋巴结转移的关系。方法 PTC患者34例,其中有颈部淋巴结转移11例,无颈部淋巴结转移23例;患者术前均行超声检查,记录肿瘤直径、边缘、纵横比、有无钙化等特征;取手术切除的PTC组织和癌旁组织各34份,采用DNA测序法检测BRAF V600E基因突变情况。比较有、无颈部淋巴结患者BRAF V600E基因突变率及超声声像特征。结果 PTC组织BRAF V600E基因突变率(52.94%)高于癌旁组织(0)(P0.05);有颈部淋巴结转移者超声声像示肿瘤钙化比率(81.82%)高于无颈部淋巴结转移者(43.48%)(P0.05),肿瘤直径[(21.36±7.54)mm]、BRAF V600E基因突变率(45.45%)、纵横比1比率(63.64%)和肿瘤边缘不光整比率(72.73%)与无颈部淋巴结转移者[(18.29±6.93)mm、56.52%、78.26%、69.57%]比较差异无统计学意义(P0.05)。结论 PTC组织BRAF V600E基因突变率较高,其与颈部淋巴结转移无明显相关性,超声声像示肿瘤钙化者可能更易出现颈部淋巴结转移。     

甲状腺乳头状癌伴桥本甲状腺炎的超声表现及其与侵袭性和BRAFV600E突变的关系

目的探讨桥本甲状腺炎(HT)是否会影响甲状腺乳头状癌(PTC)的常规超声特征表现,及其与侵袭性和BRAF~(V600E)突变的关系。方法将158例经手术证实的PTC患者分为HT组和非HT组,每组各79例,比较两组常规超声、超声造影及弹性成像特征,以及侵袭性和BRAF~(V600E)突变的差异性。应用二元Logistic回归分析超声特征与BRAF~(V600E)基因突变的相关性。结果 HT组中女性患者比例高于非HT组,差异有统计学意义(P0.05)。两组超声特征(病灶边界、纵横比、微小癌、微钙化、病灶数目、颈部淋巴结转移)、超声造影增强模式、弹性成像评分比较差异均无统计学意义。158例患者中,18例侵袭周围组织,其中HT组5例,非HT组13例;135例BRAF~(V600E)突变阳性,其中HT组63例,非HT组72例,差异均有统计学意义(均P0.05)。135例BRAF~(V600E)突变阳性病例中,两组淋巴结转移比较差异无统计学意义;侵袭周围组织情况比较差异有统计学意义(P0.05)。二元Logistic回归分析显示,158例PTC患者中,微小癌和纵横比1与BRAF~(V600E)突变阳性相关(OR=3.087、3.798,均P0.05);HT组患者中,微小癌与BRAF~(V600E)突变阳性相关(OR=3.590,P0.05)。结论 PTC合并HT好发于女性,HT不影响PTC的超声特征表现,合并HT的PTC其侵袭性及BRAF~(V600E)基因突变率均低于单纯的PTC;在PTC中,BRAF~(V600E)基因突变与微小病灶、纵横比1均相关;合并HT的PTC中BRAF~(V600E)基因突变与微小病灶相关。     

甲状腺乳头状癌BRAF V600E基因突变与临床及超声特征的相关性研究

目的 探讨甲状腺乳头状癌(PTC)BRAF V600E基因突变与临床及超声特征的相关性.方法 回顾性分析术后证实为PTC的82例患者,共96个结节的临床及超声特征.分析PTC的BRAF V600E基因突变与临床、超声特征包括2017年ACR甲状腺影像报告和数据系统(TI-RADS)之间的关系.结果 96个PTC中,突变...     

二代测序技术检测442例甲状腺乳头状癌基因突变及其临床病理学特征

目的 探讨甲状腺乳头状癌(PTC)基因改变及其与临床病理学特征的关系。方法 通过二代测序技术对442例PTC患者进行基因检测,同时收集患者的临床病理学资料。结果 (1)442例PTC患者中,423例患者检出基因突变,其中BRAF(385例)、RET(24例)、KRAS(5例)、NTRK3(3例)、NTRK1(3例);(2)BRAF基因突变仅与肿瘤最大径相关(P=0.006);(3)V600E突变丰度与性别、肿瘤最大径、肿瘤单/双侧、腺外侵犯、淋巴结转移、组织学亚型(P均<0.05)相关;(4)V600E不同突变丰度与肿瘤最大径、癌灶分布、腺外侵犯、淋巴结转移、组织学亚型(P均<0.05)相关。结论 应用NGS技术可以明确PTC各驱动基因变异的独特特征,此外BRAF基因V600E突变与多项高危的临床病理学特征相关。     

伴BRAF V600E突变的甲状腺乳头状癌超声图像特征及其淋巴结转移风险分析

目的 分析BRAF V600E突变的甲状腺乳头状癌（PTC）患者的超声图像特征，探讨其淋巴结转移风险。方法 选取我院经穿刺或手术病理组织学检查确诊的PTC患者417例，分析不同病理组织学分型PTC的BRAF V600E突变阳性率、淋巴结转移率及其超声图像特征的差异；采用多因素Logistic回归分析BRAF V600E突变与不同亚型PTC超声图像特征的关系。结果 417例PTC患者中共检出BRAF V600E突变阳性327例，其中经典亚型、滤泡亚型及高细胞亚型PTC患者中BRAF V600E突变阳性率分别为73.53%、66.67%及86.96%，高细胞亚型BRAF V600E突变阳性率高于经典亚型和滤泡亚型，差异均有统计学意义（均P<0.05）。BRAF V600E突变阳性的PTC患者中，经典亚型、滤泡亚型及高细胞亚型的颈部淋巴结转移率、结节钙化类型及边缘贴近被膜占比比较，差异均有统计学意义（均P<0.05）；各亚型BRAF V600E突变阳性患者颈部淋巴结转移率均高于突变阴性患者，差异均有统计学意义（均P<0.05）。多因素Logistic回归分析显示，BRAF ...     

BRAF基因突变在甲状腺乳头状癌中的临床意义

目的:探讨BRAF V600E基因突变与甲状腺乳头状癌的临床相关性。方法:采用PCR技术和直接测序法,对63例甲状腺乳头状癌患者和45例其他类型甲状腺疾病患者石蜡组织BRAF V600E基因突变进行检测。结果:甲状腺乳头状癌组中32例存在突变;而在其他类型甲状腺疾病组中未发现突变。BRAF V600E基因突变在甲状腺乳头状癌不同肿瘤直径组之间的比较差异以及在有无区域淋巴结转移组之间的比较差异均具有统计学意义(P0.05)。结论:BRAF V600E基因突变与甲状腺乳头状癌的发生、肿瘤的大小和区域淋巴结的转移有关。     

Electron microscopy of isolated human hepatocytes

Summary We have investigated, by scanning and transmission electron microscopy (SEM and TEM), the cell surface morphology of isolated human hepatocytes. For this purpose, liver cells were mechanically isolated from surgical or needle liver biopsies, fixed in 3% glutaraldehyde and post-fixed in 2% osmium tetroxide. In order to handle a low number of cells, a particular procedure for harvesting hepatocytes on coverslips has been developed for SEM and anin situ embedding procedure in polyethylene-embedding capsules was applied for TEM. A rough membrane exhibiting short, uniform microvilli and pores of 0.1 μ in diameter was the main feature of isolated liver cells. Furthermore, single hepatocytes showed no polarity and junctional or bile canaliculus remnants were rarely observed. However, differences in surface configuration were noted in relation to culture conditions, such as oxygen and temperature during isolation procedures. SEM, when controlled by TEM for intracellular preservation, is proposed as a reliable method for screening small quantities of hepatocyte suspensions, for intact cells and for the study of surface configuration under experimental conditions. Part of this work was made possible by a grant from the Swiss National Science Foundation (grant no. 3.849.081), Dr. A. Trevisan is a recipient of a grant fromConsiglio Nazionale delle Ricerche (CNR), Roma, Italy, for research on viral hepatitis.     

Homoeopathy for the treatment of lichen simplex chronicus: a case series

Twenty-seven patients with chronic lichen simplex involving various parts of the body were treated. Hydrocotyle was prescribed to 21 patients in different potencies (6c, 30c, 200c, 1 M, 10 M), Thuja to three patients (1 M, 10 M), Graphites (6c), Kali bich (30c) and Sulphur (200c) to one patient each during 1 year study period. Only two patients showed complete improvement with Thuja and one with Graphites. In other cases, the response was limited to partial relief of [corrected] itching.     

The development and evaluation of a nested PCR assay for detection of Neospora caninum and Hammondia heydorni in feral mouse tissues

The development of a novel nested polymerase chain reaction is described and used for detecting the presence of Neospora caninum and Hammondia heydorni DNA in DNA extracted from feral rodent tissues. A unique strategy was used for design of an assay that could be adapted for detecting DNA from more than one member of Toxoplasmatinae simultaneously with a minimal number of additional steps. The level of sensitivity described for this assay is comparable to real time-PCR and other nested PCR assays. Twenty-eight of 104 feral mice tested positive for N. caninum in at least one tissue (the brain, heart or liver) studied. In this study, eight instances are reported where the brain tested negative to N. caninum while at least one other tissue was positive. This suggests that prior studies, which screened only the brain, describe prevalence levels that are under-represented. None of 54 mouse brains tested positive for H. heydorni DNA. This suggests that mice are rarely infected by H. heydorni although this hypothesis needs to be explored further. Data obtained in the current study suggest that N. caninum is a common parasite of feral rodents which may be important in the epidemiology of the disease.     

In vitro induction of resistance to metronidazole, and analysis of mutations in rdxA and frxA genes from Helicobacter pylori isolates

In clinical Helicobacter pylori isolates, metronidazole resistance has been associated with mutations in the rdxA and frxA genes. The aim of this study was to examine the role of the rdxA and frxA genes after the in vitro induction of metronidazole resistance. A total of five suscep-tible H. pylori isolates were initially exposed to different subinhibitory metronidazole concentrations to induce in vitro resistance to metronidazole. Susceptible and resistant strains after the in vitro induction of resistance were examined to evaluate mutations of the rdxA and frxA genes by sequence analysis. After the in vitro induction of resistance, analysis revealed that two and four susceptible strains developed resistance when cultured with 0.3µg/ml and 0.6µg/ml of metronidazole, respectively. Before and after the induction of resistance, none of the susceptible strains that developed low and moderate levels of resistance presented any mutation in either of the evaluated genes, whereas strains with high-level metronidazole resistance contained a simple mutation of the frxA gene, but no specific changes in the rdxA gene. Strains with moderate-level resistance contained both single and multiple mutations of rdxA and frxA, respectively, and the low-level-metronidazole-resistant strain contained a single mutation in the frxA gene, without any significant change in the rdxA gene. In this study, the strains that developed resistance were mainly associated with mutations of the frxA gene, suggesting the possibility that inactivation of this gene could originate metronidazole resistance. The results after the in vitro induction of resistance to metronidazole suggested the presence of additional metronidazole resistance mechanisms, other than mutations of the rdxA and/or frxA genes.     

Presence of a mutation in ponA1 of Neisseria gonorrhoeae in numerous clinical samples resistant to various β-lactams and other, structurally unrelated, antimicrobials

The number of resistant strains in patients with Neisseria gonorrhoeae urethritis has been increasing, making effective treatment difficult. Chromosomally mediated penicillin-resistant N. gonorrhoeae arise through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. To understand the occurrence of penicillin resistance in patients with N. gonorrhoeae infection, we performed this study. In addition, we studied minimum inhibitory concentrations (MICs) of antimicrobials against N. gonorrhoeae strains. We measured the MICs of penicillin G, other β-lactams, and other kinds of antimicrobials against 53 clinical N. gonorrhoeae isolates from male patients with urethritis in Hyogo and Osaka, Japan. The ponA genes, encoding PBP 1 of these isolates, were sequenced. Of the 53 isolates tested, 41 strains showed some resistance to penicillin G. A mutation in the ponA (ponA1) gene was identified in 46 isolates. There was a tendency that ponA mutant (ponA1) in N. gonorrhoeae led to higher antimicrobial MICs of β-lactam antimicrobial agents (including penicillin) than those of non-ponA mutants. However, we found lower than expected MICs of penicillin and β-lactams even in ponA mutants. Therefore, we consider that detailed investigations for the further understanding of the effect of other genes, such as penC (which is reported to be related to ponA1 in achieving high-level penicillin resistance) should be our next step.     

Rapid identification of noncapsulated Streptococcus pneumoniae in nasopharyngeal samples allowing detection of co-colonization and reevaluation of prevalence

Noncapsulated pneumococci are atypical Streptococcus pneumoniae that lack a capsule and therefore do not react with any available antisera. These isolates, which are often referred as nontypeable pneumococci (NTPn), are difficult to identify as their differentiation from closely related species such as Streptococcus pseudopneumoniae and other streptococcus of the mitis group is not always straightforward. We developed a low-cost and easy assay to detect and quantify NTPn in primary samples (which may contain multiple species) obtained from nasopharyngeal swabs. The strategy is based on a multiplex polymerase chain reaction targeting lytA, cpsA, aliB-like ORF2, and 16S rDNA genes, plus a restriction fragment length polymorphism assay to differentiate typical from atypical lytA. The application of the proposed methodology to over 500 nasopharyngeal samples found that the prevalence of NTPn in colonization was 3-fold higher than that estimated by routine methods (from 2.9% to 8.6% in the study collection). The international clone Norway^NTST344 was the major clone identified.     

Community-acquired methicillin-resistant Staphylococcus aureus in Madrid, Spain: transcontinental importation and polyclonal emergence of Panton-Valentine leukocidin-positive isolates

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates producing the Panton-Valentine leukocidin (PVL) have been reported worldwide. We describe the molecular characteristics of PVL-positive CA-MRSA strains isolated in Madrid, Spain, and analyze the clinical features of patients infected with these isolates. From 2004 to 2007, we collected 13 PVL-positive MRSA isolates from patients attending to the emergency department. The isolates were genotyped by pulsed-field gel electrophoresis, SCCmec typing, agr polymorphism, and multilocus sequence typing. Susceptibility to 29 antimicrobials was determined by the broth microdilution and by the E-test methods. The isolates belonged to 3 genotypes: ST8-SCCmec IVc (n = 11), ST5-SCCmec IVa (n = 1), and ST80-SCCmec IVc (n = 1). The corresponding agr types were I, II, and III, respectively. Five isolates were resistant to tetracycline and doxycycline, and 1 was resistant to fusidic acid (ST80). The isolates were from children (n = 9) and adults (n = 4), and were associated with skin and soft tissue infections (n = 9), otitis (n = 1), and bacteremia (n = 1). Nine patients were from South America. Our results indicate the transcontinental importation and recent emergence in Spain of PVL-positive CA-MRSA strains belonging to 3 distinct lineages, including 1 predominant (ST8-SCCmec IVc).     

Antimicrobial susceptibility of respiratory tract pathogens in Japan during PROTEKT years 1–3 (1999–2002)

Data are presented on antimicrobial resistance among isolates of Streptococcus pneumoniae, Streptoco-ccus pyogenes, Haemophilus influenzae, and Moraxella catarrhalis collected in Japan during years 1–3 (1999–2002) of the Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin (PROTEKT) surveillance study. In addition to the standard panel of PROTEKT antimicrobial agents, eight other agents often used in Japan also were tested against these isolates. The majority (30%–55%) of S. pneumoniae and H. influenzae isolates were collected from patients with community-acquired pneumonia, whereas most (>70%) S. pyogenes isolates came from patients with tonsillitis/pharyngitis. Penicillin and macrolide resistance were high among isolates of S. pneumoniae, averaging 30.9%–44.5% and 77.2%–79.9%, respectively, across all centers over the 3 study years; the highest occurrences were reported among pediatric patients aged 0–2 years. The erm(B) genotype accounted for >50% of all erythromycin-resistant isolates each study year. S. pyogenes isolates were highly susceptible to most antimicrobial agents except the macrolides and tetracycline. β-Lactamase production among H. influenzae isolates range was 8.5%–9.7% per annum. A total of 9 β-lactamase-negative, ampicillin-resistant isolates were collected during the study. Almost all (>95%) M. catarrhalis isolates were β-lactamase positive each year. Telithromycin was highly active against all pathogens examined in this study during all 3 years.     

Development of a multiplex PCR for the detection of Haplosporidium nelsoni, Haplosporidium costale and Perkinsus marinus in the eastern oyster (Crassostrea virginica, Gmelin, 1971)

Haplosporidium costale (SSO), Haplosporidium nelsoni (MSX) and Perkinsus marinus (Dermo) have caused oyster mortality on the North American east coast since the 1950s. Currently, the monitoring of oyster populations for these pathogens depends on histopathology for H. nelsoni, H. costale and the Ray/Mackin assay for P. marinus. In this study we describe the development and optimization of a multiplex polymerase chain reaction (MPCR) for the detection of H. nelsoni, H. costale and P. marinus. In addition, we determine its specificity and sensitivity. The MPCR clearly detects and differentiates the protozoan pathogens. There was no cross-reactivity between these species. The MPCR was able to detect DNA from H. nelsoni as low as 10 fg and P. marinus and H. costale as low as 1 pg. The MPCR allows for the detection of H. nelsoni, H. costale and P. marinus in a single PCR reaction.     

In vitro activity of β-lactams, macrolides, telithromycin, and fluoroquinolones against clinical isolates of Streptococcus pneumoniae: correlation between drug resistance and genetic characteristics

The in vitro activity of antimicrobial agents against Streptococcus pneumoniae was determined using 16 strains of penicillin-susceptible S. pneumoniae (PSSP) and 26 strains of penicillin intermediately resistant S. pneumoniae (PISP) + penicillin-resistant S. pneumoniae (PRSP) in Japan. The minimum inhibitory concentrations (MICs) of potent antibiotics, including eight β-lactams (benzylpenicillin, ampicillin, cefotiam, cefepime, cefditoren, faropenem, panipenem, and biapenem), three macrolides (erythromycin, clarithromycin, and azithromycin), telithromycin, and three fluoroquinolones (ciprofloxacin, levofloxacin, and gatifloxacin), were determined. Twenty-three strains exhibited genetic variations at pbp1a + pbp2x + pbp2b, which are genetic-PRSP (g-PRSP). g-PISP strains accounted for 62.5% (10/16) of the PSSP strains. The existence of an abnormal pbp gene conferred not only penicillin resistance but resistance to cephems; however, panipenem and biapenem had potent in vitro efficacy against alterations. Regarding the macrolide resistance mechanisms (mefA or ermB): 16 isolates had only mefA, 18 isolates had ermB, and 2 isolates had both mefA and ermB. There was no correlation between the existence of an abnormal pbp gene and the existence of the mefA gene or the ermB gene.