多位点可变数目串联重复序列分析方法在布鲁杆菌病监测中的应用

目的 建立多位点可变数目串联重复序列分析方法(MLVA),并评价其在布鲁杆菌菌株鉴定及流行病溯源中的价值.方法 使用MLVA方法,对16株羊种菌、22株牛种菌、21株猪种菌和10株犬种菌株进行分析,使用BioNumerics(Version 5.0),对16个位点进行聚类分析,聚类方式用平均连锁聚类法(UPGMA).计算每个位点的差异指数,菌株的基因型通过MLVA2010数据库确定.结果 使用MLVA方法,通过Bruce06、Bruce08、Bruce11、Bruce12、Bruce42、Bruce43、Bruce45、Bruce55共8个位点可以区分布鲁杆菌的种;通过Bruce04、Bruce07、Bruce09、Bruce 16、Bruce30共5个位点可以对菌株进行溯源,确定菌株流行病学相关性.结论 MLVA技术是一种分辨率高且易于在省级疾病防控系统监测实验室使用的标准化分型技术,可以加强布病监测能力.     

不同VNTR位点组合用于北京基因型结核分枝杆菌基因分型的研究

目的评价不同串联重复序列（VNTR）位点组合在北京地区北京基因型结核分枝杆菌基因分型中的应用。方法采用多位点数目可变串联重复序列分析（MLVA）技术对72株北京地区北京基因型菌株的24个VNTR位点进行分析，基因多态性分析采用BioNumerics软件。结果24个位点的多态性差异较大，位点QUB-11b（HGI 0．651）、Mtub 21（HGI 0．556）和QUB-26（HGI 0．518）的多态性较高，有两个位点（MIRU2和MIRU24）则不具有多态性；不同VNTR位点组合（12位点，15位点，24位点）在北京基因型菌株中的分辨指数依次升高，分别为0．788，0．990．0．992，后两个组合的差异仅是由位点Mtub 29引起的。结论不同的VNTR位点在北京地区北京基因型菌株中的分辨力存在差异；推荐的基础VNTR15位点与Mtub 29组合可作为北京地区结核分枝杆菌分子流行病学研究的一线方法。     

辽宁省炭疽芽胞杆菌MLVA分型研究

目的 了解辽宁地区炭疽芽胞杆菌的流行特征及菌型基因特征。方法 通过多位点可变数目串联重复序列(Multiple locus variable numbers of tandem repeats analysis,MLVA)分型实验,对2001—2011年辽宁省分离到的6株炭疽芽胞杆菌分离株DNA进行检测,DNA指纹图谱使用BioNumerics 4.0 软件进行统计分析,得出聚类分析结果。结果 聚类分析发现,6株炭疽芽胞杆菌株可分为2个基因型。对于炭疽暴发而言,其可变数目串联重复序列遗传标记具有高度相似性。结论 炭疽芽胞杆菌基因组中的串联重复序列可作为炭疽芽胞杆菌基因分型的指标,在炭疽暴发事件中的病原体溯源上具有重要的意义。     

Molecular characterization of enterohemorrhagic Escherichia coli O157:H7 isolates dispersed across Japan by pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis

We identified seven distinct subtypes of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were derived from sporadic cases and outbreaks from multiple prefectures in Japan in 2005. A surveillance system utilizing pulsed-field gel electrophoresis (PFGE), PulseNet Japan, was used. Some strains showed indistinguishable PFGE patterns using another restriction enzyme (BlnI or SpeI) in each subtype of EHEC O157:H7 isolates that were routinely subtyped by the XbaI PFGE pattern. In order to examine the genotypic relatedness of these strains, we carried out a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA). By using the MLVA system, we found that three of seven subtypes of EHEC O157:H7 strains that were isolated from sporadic cases dispersed across multiple prefectures within a few months showed indistinguishable PFGE patterns and identical MLVA types. Strains belonging to the other four subtypes of EHEC O157:H7 in the PFGE analysis were further classified into different clusters of EHEC O157:H7. Therefore, compared to PFGE, MLVA showed greater discriminatory power with respect to analysis of the isolates in this study.     

Molecular epidemiological investigation of vero toxin-producing Escherichia coli (VTEC) isolated from diarrhea patients and dairy cattle

To elucidate the source and route of VTEC infection, we performed pulse field gel electrophoresis (PFGE) an 50 isolates from human diarrhea typed as serotypes O157, O111, and O26, which were very frequently isolated from patients with VTEC infection between 1986 and 1997, and 32 isolates from dairy cattle, a total of 82 isolates. The isolates were genetically analyzed based on the electrophoresis patterns of DNA, and a phylogenetic tree was prepared. The isolates were classified based on similarity > or = 89. The following results of the molecular epidemiological investigation were obtained. 1) Based on the electrophoresis patterns of DNA obtained by PFGE, 34 of the 49 O157 isolates (69.4%) were divided into groups 1-9, 15 of the 18 O111 isolates (83.3%) were divided into groups 1-3, and 12 of the 15 O26 isolates (80%) were divided into groups 1-3. Of the grouped isolates, group 8 of O157, groups 2 and 3 of O111, and group 3 of O26 included isolates from human diarrhea and dairy cattle, but the other groups included isolates from only one of the two sources. 2) With regard to regional investigation, groups 6 and 9 of O157 included human diarrhea-derived isolates from Yokohama and Ehime, and group 8 included a human diarrhea-derived isolate from Yokohama and a dairy cattle-derived isolate from Tokushima. Group 3 of O111 included a human diarrhea-derived isolate from Ehime and a dairy cattle-derived isolate from Hokkaido. Group 3 of O26 included human diarrhea-derived isolate from Ehime and dairy cattle-derived isolate from Sagamihara and Hokkaido. Since the above findings showed that although the frequency was low, isolates from human diarrhea and dairy cattle were included in the same groups, it was demonstrated that dairy cattle are closely related to the human infectious disease of the intestinal tract as a source of infection. However, classification using the PFGE method is difficult due to diversity of the electrophoresis pattern of DNA. It is necessary to investigate the classification by a combination of the PFGE method with phage typing, ribotyping, and RAPD-PCR, and to investigate more numbers of patient-derived and animal-derived isolates.     

Identification of a current hot spot of HIV type 1 transmission in Mongolia by molecular epidemiological analysis

We investigated the current molecular epidemiological status of HIV-1 in Mongolia, a country with very low incidence of HIV-1 though with rapid expansion in recent years. HIV-1 pol (1065 nt) and env (447 nt) genes were sequenced to construct phylogenetic trees. The evolutionary rates, molecular clock phylogenies, and other evolutionary parameters were estimated from heterochronous genomic sequences of HIV-1 subtype B by the Bayesian Markov chain Monte Carlo method. We obtained 41 sera from 56 reported HIV-1-positive cases as of May 2009. The main route of infection was men who have sex with men (MSM). Dominant subtypes were subtype B in 32 cases (78%) followed by subtype CRF02_AG (9.8%). The phylogenetic analysis of the pol gene identified two clusters in subtype B sequences. Cluster 1 consisted of 21 cases including MSM and other routes of infection, and cluster 2 consisted of eight MSM cases. The tree analyses demonstrated very short branch lengths in cluster 1, suggesting a surprisingly active expansion of HIV-1 transmission during a short period with the same ancestor virus. Evolutionary analysis indicated that the outbreak started around the early 2000s. This study identified a current hot spot of HIV-1 transmission and potential seed of the epidemic in Mongolia. Comprehensive preventive measures targeting this group are urgently needed.     

Imported brucellosis in Denmark: molecular identification and multiple-locus variable number tandem repeat analysis (MLVA) genotyping of the bacteria

A polymerase chain reaction was used to identify Brucella species isolated from humans in Denmark. Consecutive analysis of referred bacteria and re-examination of historical isolates identified all as Brucella melitensis. Multiple-locus variable number tandem repeat analysis (MLVA) placed the isolates in the previously defined 'East Mediterranean' B. melitensis group.     

Molecular epidemiological study of hepatitis B virus in Thailand based on the analysis of pre-S and S genes.

Aims: This study was undertaken to determine the prevalence and characteristics of hepatitis B virus (HBV) genotypes, antigen subtypes, "a" determinant variants and pre-S gene mutations circulating on a large scale in Thailand. Methods: The sequences of the Pre-S1, Pre-S2 and S regions were determined in serum samples of 147 HBsAg and HBV DNA-positive subjects who had been enrolled from the nationwide seroepidemiological survey conducted on 6213 individuals in 2004. Results: The results showed that genotypes C, B and A accounted for 87.1%, 11.6% and 1.3%, respectively. The distribution of the HBV antigen subtypes was: adr (84.4%), adw (14.2%) and ayw (1.4%). Regarding the "a" determinant, 2/43 (4.65%) and 2/104 (1.92%) samples of vaccinated and non-vaccinated subjects, respectively, displayed mutations, all ofwhich were Thr126Asn. Sequencing analysis showed the pre-S mutations in 14 (9.5%) samples, with pre-S2 deletion as the most common mutant (4.1%) followed by pre-S2 start codon mutation (2.9%), both pre-S2 deletion and start codon mutation (2.0%), and pre-S1 deletion (0.7%). The pre-S mutations were associated with older age and higher mean serum HBsAg level. Conclusion: This study demonstrated that HBV genotype/subtype C/adr and B/adw were the predominant strains circulating in Thailand. The "a" determinant variants seemed to be uncommon, and might not be attributed to vaccine-induced mutation.     

Molecular typing of methicillin-resistant Staphylococcus aureus by PCR-RFLP and its usefulness in an epidemiological study of an outbreak

A new convenient molecular typing method, simultaneous polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis, for three different genes of methicillin-resistant Staphylococcus aureus (MRSA) was evaluated using 35 isolates of MRSA and comparing results with those previously reported for sequencing-based spa typing. Twenty-nine isolates of the most frequent protein A (spa) type were discriminated into 6 different types by PCR-RFLP. In contrast, spa typing could discriminate only 1 of the 19 most frequent PCR-RFLP-type isolates. The discriminatory powers of the two methods were equal for the other isolates. These results suggest that PCR-RFLP has the advantages of both relative easiness and greater discriminatory power than spa typing. We also report the case of a suspected outbreak in which PCR-RFLP was sufficient for ruling out the possibility of an outbreak. Thus, PCR-RFLP is preferable as a preliminary screening method for epidemiological studies of nosocomial infection caused by MRSA.     

Molecular characterization of Lyme disease spirochetes (Borrelia burgdorferi sensu lato) isolated in Taiwan by restriction fragment length polymorphism analysis of 5S(rrf)-23S(rrl) intergenic spacer amplicons

We analyzed the 5S (rrf)-23S (rrl) intergenic spacer amplicon gene of Lyme disease spirochetes (Borrelia burgdorferi sensu lato) for the first time in Taiwan. The genetic identities of these Taiwan isolates (TWKM1-7) were clarified by comparing their restriction fragment length polymorphism patterns and sequence similarities of the polymerase chain reaction-amplified intergenic spacer amplicon genes with 3 major genospecies of Lyme disease spirochetes. Amplified-spacer DNAs were purified further and subjected to the cleavage by nuclease DraI or MseI. Differential fragment patterns in relation to different genospecies of Lyme disease spirochetes were observed among tested Borrelia isolates, and all of these Taiwan isolates were closely related to the genospecies of B. burgdorferi sensu stricto. The phylogenetic analysis also revealed that the sequence similarity of polymerase chain reaction-amplified spacer genes of these Taiwan isolates was highly homogeneous (95.7-100%) within the genospecies of B. burgdorferi sensu stricto and can be distinguished clearly from other genospecies of Lyme disease spirochetes with a 4.1% sequence divergence. Based on the differential fragment patterns and sequence similarity among these Taiwan isolates, the genetic identity of these Taiwan isolates should be classified into the genospecies of B. burgdorferi sensu stricto.     

Molecular epidemiological analysis of an outbreak of Campylobacter jejuni using restriction enzyme double-digestion technique for genotyping of isolates by pulsed-field gel electrophoresis

In an outbreak of gastroenteritis in elementary school students and their families in Chiba Prefecture, Japan, Campylobacter jejuni was isolated from the stools of 14 patients who developed diarrheal illness after a one-day bus trip. C. jejuni was also isolated from the stools of 3 patients not going on the bus trip. Pulsed-field gel electrophoresis (PFGE) analysis was done on 17 isolates of C. jejuni to study genetic relationships among them. PFGE profiles of isolates treated with restriction enzymes Sma I, Ksp I and Kpn I were separated into 9, 10, and 10 types, but the relationship between PFGE profiles and epidemiological profiles was unclear. Dendrograms of PFGE of isolates double-digested with both Sma I and Ksp I were typed into D1, D2, D3 and D4, and profiles compared to profiles of serotyping and flagellin typing of isolates and epidemiological profiles to evaluate genetical and epidemiological relationships. Thirteen isolates of PFGE type D1 possessed serotype G and flagellin type Al and were isolated from patients going on the bus trip. Type D2 isolated from a student going on the bus trip and type D3 isolates from two students not going on the bus trip had serotype B and flagellin type A2. C. jejuni of PFGE type D4, serotype UT, and flagellin type A3 was also isolated from a student not going on the trip. Our results show that at least two outbreaks of C. jejuni occurred simultaneously in people related to the school. Restriction enzyme double-digestion PFGE was thus useful in the molecular epidemiological analysis of the C. jejuni outbreak.     

Identification of susceptibility loci for Type 1 (insulin-dependent) diabetes by trans-racial gene mapping

Summary A major component of inherited susceptibility to Type 1 (insulin-dependent) diabetes mellitus has been mapped to the major histocompatibility complex. Certain gene alleles in this region determine susceptibility and resistance to the disease. Mapping of susceptibility is hindered by the limitations of conventional tissue typing techniques, and by strong linkage disequilibrium within this part of the genome. Recombinant DNA technology and trans-racial studies have been used to allow finer mapping of genetic predisposition to Type 1 diabetes. These techniques have localised alleles encoding susceptibility and resistance to the DQ region. Other alleles determining disease susceptibility remain poorly localised.     

Repeated measures study of human herpesvirus 8 (HHV-8) DNA and antibodies in men seropositive for both HHV-8 and HIV

OBJECTIVE: To study the natural history and pathogenesis of human herpesvirus 8 (HHV-8) infection in HHV-8-seropositive, immunosuppressed men. DESIGN: Longitudinal study of 87 HHV-8- and HIV-seropositive men [42 with Kaposi's sarcoma (KS)] during four visits over a 2 month period. METHODS:: Patients provided oral fluid and blood. HHV-8 antibody titers were measured with peptide-based enzyme-linked immunosorbent assays (ELISA) for ORF65 and K8.1; HHV-8 DNA was detected with polymerase chain reaction ELISA. RESULTS: HHV-8 DNA was present in oral fluid or peripheral blood mononuclear cells (PBMC) at one or more of the four visits in 71% of men with KS and 56% of men without KS. The strongest correlate of HHV-8 DNA in PBMC was the presence of KS [odds ratio (OR), 8.7; 95% confidence interval (CI), 3.4-22]. Detection of HHV-8 DNA in oral fluid or PBMC was often intermittent, but individuals who shed virus at one time point were more likely to shed at other times. Some men had incomplete epitope recognition in their anti-HHV-8 antibody response. High antibody titers were associated with the absence of circulating HHV-8, particularly for the ORF65 seroassay (OR, 0.16; 95% CI, 0.05-0.51). CONCLUSIONS: Among HHV-8 seropositive men, circulating virus is common even in the absence of disease. The link between KS and HHV-8 DNA in PBMC suggests that anti-herpes drugs may impede KS development or progression. Seroassays should target multiple epitopes to achieve maximal sensitivity. HHV-8 replication may be limited by high antibody titers or other immune function for which antibodies are a marker.     

Rapid epidemiological mapping of onchocerciasis (REMO): its application by the African Programme for Onchocerciasis Control (APOC)

One of the fundamental challenges that the African Programme for Onchocerciasis Control (APOC) has had to face is how to identify the endemic communities where its mass ivermectin-treatment operations are to be carried out in conformity with its stated objective of targetting the most highly endemic, affected and at-risk populations. This it has done by adopting a technique, known as the rapid epidemiological mapping of onchocerciasis (REMO), that provides data on the distribution and prevalence of onchocerciasis. Integration of the REMO data into a geographical information system (GIS) enables delineation of zones of various levels of endemicity, and this is an important step in the planning process for onchocerciasis control. Zones are included in (or excluded from) the APOC-funded programme of community-directed treatment with ivermectin (CDTI), depending on whether or not their levels of onchocercal endemicity reach the threshold set by APOC. This review describes the application of the REMO/GIS technique by APOC in its operations, and identifies the remaining related challenges.     

Poly(gamma-D-glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of Bacillus anthracis: a potential addition to the anthrax vaccine

Both the protective antigen (PA) and the poly(gamma-d-glutamic acid) capsule (gamma dPGA) are essential for the virulence of Bacillus anthracis. A critical level of vaccine-induced IgG anti-PA confers immunity to anthrax, but there is no information about the protective action of IgG anti-gamma dPGA. Because the number of spores presented by bioterrorists might be greater than encountered in nature, we sought to induce capsular antibodies to expand the immunity conferred by available anthrax vaccines. The nonimmunogenic gamma dPGA or corresponding synthetic peptides were bound to BSA, recombinant B. anthracis PA (rPA), or recombinant Pseudomonas aeruginosa exotoxin A (rEPA). To identify the optimal construct, conjugates of B. anthracis gamma dPGA, Bacillus pumilus gamma dLPGA, and peptides of varying lengths (5-, 10-, or 20-mers), of the d or l configuration with active groups at the N or C termini, were bound at 5-32 mol per protein. The conjugates were characterized by physico-chemical and immunological assays, including GLC-MS and matrix-assisted laser desorption ionization time-of-flight spectrometry, and immunogenicity in 5- to 6-week-old mice. IgG anti-gamma dPGA and antiprotein were measured by ELISA. The highest levels of IgG anti-gamma dPGA were elicited by decamers of gamma dPGA at 10 -20 mol per protein bound to the N- or C-terminal end. High IgG anti-gamma dPGA levels were elicited by two injections of 2.5 microg of gamma dPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies. rPA was the most effective carrier. Anti-gamma dPGA induced opsonophagocytic killing of B. anthracis tox-, cap+. gamma dPGA conjugates may enhance the protection conferred by PA alone. gamma dPGA-rPA conjugates induced both anti-PA and anti-gamma dPGA.     

A clinical and epidemiological study of "ornithosis" caused by Chlamydia psittaci and Chlamydia pneumoniae (strain TWAR)

Ornithosis is a notifiable disease in Sweden since 1954. In 1981 and 1982 a sharp increase in the number of notifications occurred. Since then the number has declined but is still high. A changed epidemiology characterized by no history of bird contact and no common source, raised the suspicion of a new agent. Serological data now suggest that the epidemic was to a substantial part due to Chlamydia pneumoniae (strain TWAR) (48% of the patients during 1981-1982 compared to 9% during 1984-1987). During recent years TWAR infections have thus become uncommon but reappearance can be expected in the near future. The clinical picture as well as the complications appear to be very similar in infections caused by C. pneumoniae and C. psittaci.