血流感染肺炎克雷伯菌临床特征、毒力基因及分子流行病学研究

摘要：目的 探讨血流感染肺炎克雷伯菌(KPN)的临床及分子生物学特征。方法 收集2019年1月-2020年12月,血流感染患者血液分离的KPN。应用Microflex LT型质谱仪进行菌种鉴定,Vitek-2分析仪进行药敏试验,结果判定参照CLSI 2019版标准。检测KPN多位点序列分型、荚膜分型及毒力因子等。应用全基因组测序技术分析其中碳青霉烯类耐药KPN(CRKP)分子生物学特征。结果 2年内共有72株KPN纳入研究,根据感染类型分为医院感染(HABSI)组(39株,54.2%)及社区感染(COBSI)组(33株,45.8%)。在入住ICU＞10d、侵袭性诊疗、使用碳青霉烯类药物和死亡或放弃治疗方面,HABSI组患者的比例高于COBSI组(P<0.05)。HABSI组对常用抗菌药物的耐药率高于COBSI组。HABSI组菌株中共检出19株CRKP(26.4%),94.7%的CRKP携带blaKPC-2,主要为K64-ST11型,且存在高度亲缘关系,所有K64-ST11型CRKP均携带IncHI1B型质粒。结论 血流感染患者血液分离的CRKP呈现多重耐药,K64-ST11型为其主要分子型别,携带多种质粒介导的毒力因子,且存在医院内感染的克隆播撒。     

Antimicrobial susceptibility and molecular epidemiology of Streptococcus pneumoniae isolated from Shanghai, China

In this study, the antimicrobial resistance profiles of pneumococci isolated from respiratory specimens of patients from Shanghai, China, in 2004 and 2005 are described. Non-susceptible rates to penicillin and erythromycin among paediatric isolates (n=122) were 63.1% and 94.3%, respectively, whilst those of adult isolates (n=39) were 20.5% and 69.2%, respectively (P<0.0001 and P<0.0002). Nineteen serotypes were identified among 103 tested strains, 73.8% of which belonged to 19F, 14, 23F, 6B and 19A. The erm(B) gene was detected in 51 erythromycin-resistant strains (52.6%), the mef(E) gene in 5 strains (5.2%) and both erm(B) and mef(E) in 41 strains (42.3%). Among 45 sequence types (STs) determined by multilocus sequence typing (MLST) in these 103 isolates, 25 STs were new assignments and 9 STs contained 10 new alleles. On the other hand, 46 (68.7%) of 67 penicillin-non-susceptible S. pneumoniae and 51 (52.6%) of 97 macrolide-resistant S. pneumoniae were characterised as belonging to four international resistant clonal complexes, Taiwan(19F)-14, Spain(23F)-1, Spain(6B)-2 and Taiwan(23F)-15. Our findings indicate that the spread of international resistant clones played a predominant role in the emergence and increase of resistance in Shanghai. Conjugate vaccination may be a promising method to prevent the increase in pneumococcal resistance.     

Cluster analysis of genetic and epidemiological data in molecular epidemiology

Current molecular epidemiological studies of complex diseases include a large number of genetic and epidemiological variables. Clustering approaches are a useful tool to detect patterns in data sets and generate hypothesis regarding potential relationships in complex data situations. In this article similarity coefficients are presented for a hierarchical cluster analysis of single-nucleotide polymorphisms (SNPs) and epidemiological data to gain insight into the relationship of variables and detect potential differences between diseased and control individuals in case-control studies. This approach was applied to two subsets of data from the GENICA study of sporadic breast cancer, a molecular epidemiological population-based case-control study conducted in the greater Bonn region between 2000 and 2004. Separate cluster analyses for cases and controls using flexible matching coefficients for SNPs, Pearson's corrected coefficient of contingency for categorical epidemiological variables, and Spearman's correlation coefficient for quantitative epidemiological variables as measures of similarity revealed small subgroups of SNPs usually of the same gene, as well as clusters of genetic and of epidemiological variables with minor differences between cases and controls. In addition to recent and well-known findings, the joint cluster analysis of SNPs and epidemiological variables provides further insight into the relationship of these variables.     

ST37型艰难梭菌分子流行病学研究

目的 应用全基因组分析方法对艰难梭菌感染可能的暴发流行进行识别和调查,为艰难梭菌感染防控提供可靠的分子流行病学基础。方法 对8株ST37型艰难梭菌进行第二代高通量测序,并完成序列的拼接和注释。通过对其核心基因组进行SNP分析,根据SNP数量以及相关临床资料分析有无院内暴发流行。同时将Genbank中公布了全基因组序列的艰难梭菌,进行MLST分析,对其分析结果为ST37的菌株与本研究中的8株菌株进行SNP分析比较,了解这8株菌的可能来源。结果 以最早收集的菌株WCHCD770作为参照,其他7株菌株的SNP值,最大为59,最小为38,提示它们并不是近期发生的传播事件。而这8株菌两两相互进行SNP计算,其中WCHCD1577、WCHCD1641仅为10,提示它们可能来源于一个克隆,可能存在院内传播。通过分析比较这8株菌与Genbank中的其他ST37型艰难梭菌发现,它们的致病决定区(PaLoc)序列完全相同,证实了ST37型艰难梭菌的PaLoc在世界范围克隆传播,同时对它们的SNP比较分析,发现WCHCD1577、WCHCD1641、WCHCD1216、WCHCD109、WCHCD159与2...     

A PCR method for molecular epidemiology of Plasmodium falciparum Msp-1

Merozoite surface protein-1 (MSP-1) of Plasmodium falciparum is a strong malaria vaccine candidate. However, MSP-1 exhibits extensive antigenic polymorphism, an issue which may compromise the development of effective vaccine based on this molecule. Since polymorphic nature of MSP-1 has not been fully understood in endemic areas of malaria, variation of the MSP-1 gene (Msp-1) must be studied in detail in natural parasite populations. Here, a PCR-based method for determination of P. falciparum Msp-1 haplotype is described, which can detect up to 24 different haplotypes per infected person. The method can be applied to various purposes of molecular epidemiology of not only Msp-1 haplotype but the genetic structure of P. falciparum populations.     

Surveillance based on molecular epidemiology for Haemophilus influenzae Isolates in Gifu Prefecture

We analyzed Haemophilus influenzae isolates in Gifu prefecture between May 2003 and August 2003. We conducted molecular-level epidemiological studies for 313 strains using PCR to identify resistant genes in H. influenzae. Our four sets of primers are as follows: (i) p6 gene of P6 membrane protein, (ii) TEM-1 type beta-lactamase gene (bla), (iii) normal PBP 3 gene (ftsl), and (iv) mutational ftsl gene detected in beta-lactamase-nonproducing ampicillin (ABPC) resistant H. influenzae (BLNAR). H. influenzae strains were classified into 6 types based on PCR: (i) beta-lactamase-nonproducing ABPC-susceptible strains (BLNAS; n = 85) with no any resistant genes, (ii) TEM-1 type beta-lactamase-producing ABPC resistant strains (BLPAR; n = 6), (iii) beta-lactamase-nonproducing and low-level ABPC-resistant strains (Low-BLNAR; n = 77) possessing Asn-526 --> Lys-526 amino acid substitution, (iv) BLNAR strains (n = 138) possessing Asn-526 --> Lys-526 and 3 amino acids substitutions detected around the Ser-Ser-Asn conserved motif, (v) beta-lactamase-producing amoxicillin-clavulanate resistant strains (BLPACR-I; n = 3) possessing TEM-1 and Low-BLNAR resistant genes, and (vi) beta-lactamase-producing amoxicillin-clavulanate resistant strains (BLPACR-II; n = 4) possessing TEM-1 and BLNAR resistant genes. Amoxicillin (AMPC) MIC90s in Low-BLNAR was 4 microg/mL and in BLNAR was 16 microg/mL. In oral cephalosporins, cefditoren MIC90 was the most excellent with 0.5 microg/mL against BLNAR. The prevalence of H. influenzae type b isolates in Matsubara Otorhinolaryngology Clinic was 66.7%. Selection of appropriate antimicrobial agents should be performed to prevent resistant microorganisms. Also, the vaccination for H. influenzae type b would be strongly recommended in near future.     

Molecular epidemiology of clinically significant antibiotic resistance genes

Antimicrobials were first introduced into medical practice a little over 60 years ago and since that time resistant strains of bacteria have arisen in response to the selective pressure of their use. This review uses the paradigm of the evolution and spread of beta-lactamases and in particular beta-lactamases active against antimicrobials used to treat Gram-negative infections. The emergence and evolution particularly of CTX-M extended-spectrum beta-lactamases (ESBLs) is described together with the molecular mechanisms responsible for both primary mutation and horizontal gene transfer. Reference is also made to other significant antibiotic resistance genes, resistance mechanisms in Gram-negative bacteria, such as carbepenamases, and plasmid-mediated fluoroquinolone resistance. The pathogen Staphylococcus aureus is reviewed in detail as an example of a highly successful Gram-positive bacterial pathogen that has acquired and developed resistance to a wide range of antimicrobials. The role of selective pressures in the environment as well as the medical use of antimicrobials together with the interplay of various genetic mechanisms for horizontal gene transfer are considered in the concluding part of this review.     

肺炎克雷伯菌临床分离株的耐药性与分子流行病学分析

目的了解我院住院患者临床肺炎克雷伯菌分离株的耐药性与分子流行特征,为临床抗感染及合理用药提供依据。方法收集2018年4-12月我院住院病人肺炎克雷伯菌临床分离株,采用纸片扩散法或自动化仪器法按统一方案进行抗菌药物敏感性试验;聚合酶链反应(PCR)法检测耐药基因;脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)技术对菌株进行同源性分析和分型。结果共分离到肺炎克雷伯菌211株,对第3代头孢(头孢噻肟)和第四代头孢(头孢吡肟)的耐药率分别为54.5%和44.1%,对碳青霉烯类抗菌药物亚胺培南和美罗培南的耐药率为32.7%和34.1%;其中107株(107/211,50.6%)菌株分离自ICU病房,56株(56/107,52.3%)为碳青霉烯类耐药肺炎克雷伯菌(CR-KP),MLST分型显示以ST11为主,PFGE分型结果主要为A型和F型两大聚类。结论肺炎克雷伯菌耐药率高,特别是分离自ICU病房的菌株耐药情况尤为严重,且携带KPC-2基因的ST11克隆株在ICU病房流行传播。     

The molecular epidemiology of pain: a new discipline for drug discovery

Recent candidate gene studies have identified and replicated the first associations between several common polymorphisms and pain severity in humans. Moreover, human studies in twins suggest high heritability for responses to experimental pain stimuli. Human genome-wide association studies of pain phenotypes might identify novel analgesic targets, help to prioritize research among current targets, and increase the likelihood of success for analgesic candidates emerging from animal studies. However, clinical research in pain has largely focused on small neurophysiology-based studies, so expansion of epidemiological understanding will be essential to the success of genetic or proteomic dissection of complex pain disorders. This Perspective outlines how methods of molecular epidemiology, proved effective in the study of other diseases, can enhance the returns from human genomic studies and expedite the development of new drugs to prevent or treat pain.     

Cervical cancer and CYP2E1 polymorphisms: implications for molecular epidemiology

Introduction Besides human papillomavirus (HPV) infection, several cofactors are considered important for the development of cervical cancer (CC). Among these, tobacco smoke, other sexually transmitted diseases, inflammation and nutritional factors have been intensively described. CYP2E1 polymorphisms have been associated with the metabolization of several carcinogens, some of them considered risk factors for CC development, such as tobacco smoke. The aim of this study was to evaluate the role of CYP2E1 polymorphisms in the susceptibility to cervical cancer in a Portuguese population. Patients and methods The genotypic analysis was performed with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology, using peripheral blood samples of 454 individuals: 122 presented invasive squamous cell carcinoma (ICC), 59 presented squamous intraepithelial lesions (SIL), and the control population was composed of 274 healthy individuals. Results Concerning the DraI polymorphism, we observed a decreased risk for the development of squamous cervical lesions in the presence of the C allele [odds ratio (OR)=0.600; 0.378<OR<0.952; p=0.029]. In the stratification of the analysis according to the mean age, we observed an increased risk for the development of SIL, for women older than 39 years of age, in the presence of the D allele (OR=0.087; 0.012<OR<0.651; p=0.003). Regarding the RsaI polymorphism, we did not find any significant differences. Conclusion The decreased risk observed for the development of SIL and not ICC in the presence of the D allele may indicate that CYP2E1 interferes with the initial steps of the carcinogenic process, probably due to its involvement in the action of immunological mediators, expressed during cervical inflammation. These aspects may help to define new therapeutic strategies for chemoprevention.     

B群脑膜炎球菌的分子流行病学与疫苗设计

脑膜炎球菌是一种严格的人类病原菌,主要引起发病率和病死率均较高的流行性脑脊髓膜炎和败血症.过去对脑膜炎球菌的研究主要以血清学实验为基础.随着分子生物学技术的发展,B群脑膜炎球菌的分子流行病学研究取得了极大的进展,获得的研究成果为疫苗开发与评价提供了重要的依据.此文对B群脑膜炎球菌的分子流行病学及其疫苗设计进行简要论述.     

结核病传播链分子流行病学研究的病例选择方法初探

目的 对以患者居住地为基础的结核病传播链分子流行病学研究的病例选择方法进行理论性评估.方法 在本院建立的广州地区分枝杆菌菌株库中提取9 234株结核分枝杆菌相应的患者信息资料,以患者居住地假定其间传播关系的有无,分析每条传播链中患者发病时间间隔及年龄组合情况.结果 以县-乡镇-村-组等农村区划、市-区-社区-街道等城市区划为路线,发现387条假定结核病传播链,以2-5病例传播链为主,分别为210、100、35、23条,其它则包括6、7、8、1l、12病例传播链,分别为6、7、4、1、l条.从传播链中患者居住地看,农村地区以同村为主,占农村区划传播链的89.39％(59/66),城市地区以同街道为主,占城市区划传播链的89.10％(286/321);传播链中患者发病时间间隔在半年、1年、1年半、2年、2年半、3年内及大于3年的传播条数分另为131、81,59、59、50、6和1;按少年(＜ 15岁)、青年(15～44岁)、中年(45～64岁)、老年(≥65岁)4个年龄段划分,不同性别跨1年龄段、同性别同年龄段、不同性别跨2年龄段、不同性别同年龄段、同性别跨1年龄段、同性别跨2年龄段、不同性别跨3年龄段的传播链条数分别为94、69、69、67、59、28、l条.结论 以患者居住地为基础的传播链分析所获得的结核病传播链分子流行病学研究的病例比较符合现行结核病传播理论,在结核病传播链分子流行病学研究中切实可行.     

Successful treatment of imported mucosal Leishmania infantum leishmaniasis with miltefosine after severe hypokalemia under meglumine antimoniate treatment

Old World mucosal leishmaniasis is a rare but regularly reported disease in Southern Europe. We report the case of a 64-year-old woman who developed severe hypokalemia under meglumine antimoniate treatment and was successfully treated under second line therapy with miltefosine.     

Evaluation of vaccinal effectiveness of preparations containing membrane antigens of Leishmania (L.) amazonensis in experimental cutaneous leishmaniasis model

American tegumentary leishmaniasis (ATL) is considered a neglected disease, for which an effective vaccine or an efficient diagnosis is not yet available and whose chemotherapeutic arsenal is threatened by the emergence of resistance by etiological agents such as Leishmania amazonensis. ATL is endemic in poor countries and has a high incidence in Brazil. Vaccines developed from native parasite fractions have led to the identification of defined antigenic subunits and the development of vaccine adjuvant technology. The purpose of the present study was to develop and compare preparations based on membrane antigens from L. amazonensis, as a biotechnological prototype for the immunoprophylaxis of the disease in a murine experimental model. For this purpose, batches of biodegradable polymeric micro/nanoparticles were produced, characterized and compared with other parasite's antigens in solution. All preparations containing membrane antigens presented low toxicity on murine macrophages. The in vivo evaluation of immunization efficacy was performed against a challenge with L. amazonensis, along with an evaluation of the immune response profile generated in BALB/C mice. The animals were followed for sample processing and quantification of serum-specific cytokines, nitrites and antibodies. The sera of animals immunized with the non-encapsulated antigen formulations showed higher intensities of nitrites and total IgGs. This approach evidenced the importance of the biological studies involving the immune response of the host against the parasite being interconnected and related to the subfractionation of its proteins in the search for more effective vaccine candidates.     

Functional cloning as a means to identify Leishmania genes involved in drug resistance

Resistance to anti-leishmanial drugs is a mounting problem in high-endemicity regions of South Asia and, potentially, in the context of HIV-Leishmania coinfections in Southern Europe. The molecular basis for clinical drug resistance is still largely unknown. It is important, however, to identify all relevant drug resistance markers for further drug development and for epidemiological surveys. An elegant and powerful method to identify such drug resistance markers without bias is functional cloning, using cosmid-based genomic DNA libraries. This review discusses the merits and caveats of this approach.     

Suggestions for the nomenclature of human alleles: relevance to ecogenetics, pharmacogenetics and molecular epidemiology

The current number of 9422 symbols for human gene names (http://www.gene.ucl.ac.uk/nomenclature/) is expected to increase 7- to 15-fold over the next 2 years. In and around each gene, a tremendous degree of single-nucleotide polymorphism (SNP) heterogeneity is now realized to exist. This review is intended to be visionary, to point out some of the enormously complex nomenclature issues that we face, and to offer some reasonable solutions to these issues. For example, I believe that a 'gene' should be defined as that region from the furthest 5'-ward enhancer to at least 150 bases downstream of the last exon. Just as established rules are critically important for the systematic naming of all new genes, standardized nomenclature rules for the naming of allelic variants are also desperately needed. The evolving consensus for naming the alleles of all human genes (ideally based on evolutionarily diverging haplotype patterns) is described herein. Because of the anticipated explosion in finding new genes and allelic variants due to high-throughput resequencing and DNA-chip technologies, this excess of new knowledge will undoubtedly overwhelm their publication by scientific journals alone. I suggest that the best approach to this staggering 'information overload' is to place the data on appropriate web sites--with numerous links between sites, and frequent updates of all information--so that colleagues in all fields of medical and genetic research can remain knowledgeable. Examples of successful web sites to date include those for the cytochrome P450 (CYP) genes and human CYP alleles, UDP glycosyltransferase (UGT) genes and human alleles, human N-acetylaminotransferase (NAT2, NAT1) alleles, and aldehyde dehydrogenase (ALDH) genes and human alleles. Many more web sites will be necessary. For each site, the webmaster will need to be responsible, accurate, energetic, highly organized, and keen to keep the site current. I believe that interactive discussions on these sites should be encouraged, and advisory committees must be willing to check frequently to ensure that all new information is accurate. Lastly, for the field of molecular epidemiology, the importance of correlating an informative genotype with an unequivocal phenotype is emphasized, and the emerging realization that racial and ethnic groups are highly admixed is summarized and updated.