Distribution of genotypes and antibiotic resistance genes among invasive Streptococcus agalactiae (group B streptococcus) isolates from Australasian patients belonging to different age groups.

Serotype distribution and antibiotic resistance (AR) among group B streptococci (GBS) affect GBS disease prevention strategies, but vary among patient groups. A multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay was used to compare the distributions of GBS serotypes, serotype III subtypes and AR-associated genes among 666 invasive isolates from 663 patients, divided into five age groups: infants, early-onset (EO; 0-6 days) and late-onset (LO; 7-90 days); children (aged 3 months to 14 years); women of childbearing age (WCBA; aged 15-45 years); and other adults (males aged >15 years; females aged >45 years). Serotypes Ia and V and serosubtype III-1 accounted for 60% of infections. Serosubtype III-2, which corresponds to a virulent clone belonging to sequence type (ST)17, was relatively uncommon overall (7%), but was associated strongly with LO infant infections, in which it was significantly more common than in adult infections (25/104 (24%) vs. 9/392 (2%), p <0.0001) or in EO infections (25/104 (24%) vs. 14/155 (9%), p <0.005). Erythromycin resistance genes were found in 8% of all isolates (ermB 3%, ermA 2.5% and mefA/E 2%), in 11-15% of isolates of serotypes II and V and subtype III-1, but in none of the isolates of serosubtype III-2 (III-2, 0/49 vs. all others, 54/618 (9%), p <0.04). In summary, the virulent serosubtype III-2 was associated strongly with LO infant GBS infection, but was less likely than other serotypes or serosubtype III-1 to carry AR genes.     

Use of a multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay for the rapid identification of bacterial pathogens.

The aim of this study was to develop a sensitive and reliable method for the molecular identification of pathogenic bacteria. A multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay was developed and evaluated for the rapid identification of 24 systemic and respiratory bacterial pathogens in routine diagnosis. All species-specific probes designed for the RLB hybridised with amplified DNA only from the corresponding species. Sensitivity limits of the mPCR/RLB assay varied among the 24 target organisms from 0.05 pg to 0.5 ng of genomic DNA. The sensitivity of the assay was 2 x 10(2) CFU/mL for Streptococcus pneumoniae and 6 x 10(2) CFU/mL for Escherichia coli. The specificity of each probe was tested against 24 species. There were no cross-reactions among any of the 43 probes. The mPCR/RLB assay appeared to be a useful alternative tool for the molecular identification of common pathogens.     

Multiplex PCR for identification of herpes virus infections in adolescents

The aim of the study was to develop a multiplex PCR (mPCR) for a rapid and simultaneous detection of herpes simplex 1 (HSV-1), herpes simplex 2 (HSV-2), and human cytomegalovirus (HCMV) DNA in squamous oral cells obtained from adolescents. Accuracy of the method was tested in a group of 513 adolescents, almost 11% of subjects were positive for infection with herpes viruses. Correlations with gender, age, and place of residence were sought. A similar incidence of HSV-2 and HCMV was found (4.3% and 5.4%, respectively) and the incidence of HSV-1 was the lowest (1%) in the study group. Conversely to HSV-2, HCMV was detected mostly in the youngest individuals. The same occurrence of all viruses was observed in boys and girls. The mPCR method described is suggested as a useful tool for epidemiologic studies of active herpes infections.     

4例假肥大型肌营养不良患儿的基因检测及临床分析

目的假肥大型肌营养不良(DMD)基因缺失的检测,为进一步产前基因诊断提供可靠的方法。方法采用18对引物的多重链式聚合酶反应(mPCR)检测4例DMD患儿的基因突变类型。结果4例患儿中2例检测出基因缺失,其中1例患儿为50、51外显子缺失,另1例患儿为43外显子缺失。结论mPCR在DMD基因缺失诊断中具有快速、准确、经济、简便的特点,便于临床推广。     

Molecular characterization of virulence and antimicrobial resistance profile of Shigella species isolated from children with moderate to severe diarrhea in northeastern Brazil

Molecular characterization of virulence and antimicrobial resistance profiles were determined for Shigella species isolated from children with diarrhea in Fortaleza, Brazil. Fecal specimens were collected along with socioeconomic and clinical data from children with moderate to severe diarrhea requiring emergency care. Shigella spp. were isolated by standard microbiological techniques, and we developed 4 multiplex polymerase chain reaction assays to detect 16 virulence-related genes (VRGs). Antimicrobial susceptibility tests were performed using disk diffusion assays. S. flexneri and S. sonnei were the predominant serogroups. S. flexneri was associated with low monthly incomes; more severe disease; higher number of VRGs; and presence of pic, set, and sepA genes. The SepA gene was associated with more intense abdominal pain. S. flexneri was correlated with resistance to ampicillin and chloramphenicol, whereas S. sonnei was associated with resistance to azithromycin. Strains harboring higher numbers of VRGs were associated with resistance to more antimicrobials. We highlight the correlation between presence of S. flexneri and sepA, and increased virulence and suggest a link to socioeconomic change in northeastern Brazil. Additionally, antimicrobial resistance was associated with serogroup specificity in Shigella spp. and increased bacterial VRGs.     

Progress of targeted and immunotherapy for hepatocellular carcinoma and the application of next-generation sequencing

Hepatocellular carcinoma (HCC), leading cancer worldwide, has a high degree of genetic heterogeneity; next-generation sequencing (NGS) technology has contributed significantly to the discovery of driving genes as well as high-frequency mutations in HCC. The detection of gene alterations may allow us to predict prognosis and adverse drug reactions for individuals, paving the way for personalized medicine in HCC patients. In this review, we summarized the common systemic therapy regimens for HCC and the predictive efficacy of genetic biomarkers on the prognosis of patients under these treatments. Finally, we put forward a future perspective on the potential of NGS technology for the guidance of targeted therapy and immunotherapy in HCC.     

反向线性点杂交技术在肺炎链球菌分型中的应用

目的 探讨反向线性点杂交技术在肺炎链球菌分型中的应用.方法 对临床诊断为肺炎的16例5岁以下患儿,从呼吸道吸取物、血和胸水培养分离获得的30株肺炎链球菌菌株应用反向线性点杂交技术进行分型,并与传统的血清学分型方法-夹膜肿胀实验进行比较.结果 反向线性点杂交技术和血清学方法对30株临床分离获得的肺炎链球菌菌株的分型结果完全一致,其分布情况为:血清型19A(n=9),14(n=5),23F(n=4),19F(n=4),6B(n=3),7F(n=2),15B(n=2),9V(n=1).16例肺炎患儿的肺炎链球菌血清型分布情况为血清型19A、14、23F及19F各来自3例患儿,血清型6B、7F、15B及19V各来自1例患儿.其中10例(62.5％)肺炎患儿分离获得的肺炎链球菌菌株涵盖在7价肺炎链球菌结合疫苗中,所有患儿获得的菌株均包含在23价多糖疫苗中.结论 对于肺炎链球菌,反向线性点杂交技术是一种可靠的分子生物学分型方法;在本组研究中,7价肺炎链球菌结合疫苗的涵盖率为62.5％,所有菌株均包含在23价多糖疫苗中.     

A multiplex PCR for rapid and simultaneous detection of porcine circovirus type 2, porcine parvovirus,porcine pseudorabies virus,and porcine reproductive and respiratory syndrome virus in clinical specimens

A multiplex PCR (mPCR) assay was developed and evaluated for its ability to simultaneously detect multiple viral infections of swine. Specific primers were designed for each of the following four DNA or RNA viruses: porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each target produced a specific amplicon with a size of 353 bp (PCV2), 271 bp (PPV), 194 bp (PRV), or 434 bp (PRRSV). The assay was sensitive and specific in detecting each target agent in composite cell cultures and clinical specimens. Results from mPCR were confirmed by PCR for individual viruses and by virus isolation. In conclusion, the mPCR has the potential to be useful for routine molecular diagnosis and epidemiology.     

福氏志贺菌临床分离株毒力基因的研究

目的研究在一种快速、特异的mPCR方法在检出福氏志贺菌的同时对其毒力进行监测。方法选取福氏志贺菌三种毒力基因ipaH,ial,set1B作为扩增目标,在同一mPCR体系中对86株福氏志贺菌临床分离株进行了检测,并选取12株进行了噬斑形成试验,观察了扩增产物不同的福氏志贺菌对Hela细胞的毒力作用。结果86株福氏志贺菌临床分离株均检测到ipaH基因,阳性率为100%;45株检测到ial基因,阳性率为52%;69株检测到set1B基因,阳性率为80%。噬斑形成试验证明,mPCR结果不同的菌株对Hela细胞的感染能力存在明显差异。结论应用上述mPCR体系在检出福氏志贺菌的同时能够对菌株的毒力进行初步判别,对于临床诊断及治疗具有重要意义。     

DMD的产前基因诊断小结

ＤＭＤ(假性肥大型进行性肌营养不良症 )是具严重危害的Ｘ染色体连锁隐性遗传病 ,男性活产婴儿的发病率为 1/ 35 0 0 ,一般 3～ 6岁发病 ,进行性加重 ,12岁左右即不能行走 ,约 2 0岁夭折 ,无有效治疗方法 ,其防治的基本措施是对ＤＭＤ高危孕妇进行胎儿产前基因诊断 ,不让有病患儿出生。目前通用的产前基因诊断步骤为 :用ｍＰＣＲ(多重聚合酶链反应 )筛测先证者患儿的基因变异类型 ,对缺失型家系可用ｍＰＣＲ分析羊水ＤＮＡ是否缺失与先证者同样的基因片段 ;而对非缺失家系 ,则采用ＳＴＲ(短串联重复系列 )多态的单体型连锁分析。现将笔者对3…