Evidence that the gonococcal porA pseudogene is present in a broad range of Neisseria gonorrhoeae strains; suitability as a diagnostic target

AIMS: The primary aim of the study was to determine if the gonococcal porA pseudogene is a stable sequence target for the detection of Neisseria gonorrhoeae by PCR. METHODS: A total of 240 gonococcal strains from various geographic locations were tested by porA pseudogene PCR. In addition, porA pseudogene PCR positivity rates were compared with established gonococcal assays in three Australian states. RESULTS: All N. gonorrhoeae isolates provided positive results in the porA pseudogene PCR. Positivity rates compared favourably with established gonococcal assays, with increased N. gonorrhoeae detection in the Northern Territory and Western Australia. CONCLUSIONS: The results of this multicentre study provide further evidence that the porA pseudogene is highly conserved across a diverse range N. gonorrhoeae strains and is a suitable PCR target for routine detection of N. gonorrhoeae.     

淋病球菌单克隆抗体的制备和特性的研究

应用杂交瘤技术筛选出２株淋球菌特异性单克隆抗体Ｎｇ４（ＩｇＧ２ａ）和Ｎｇ１８（ＩｇＧ３）有可能用于淋病的实验诊断．２株单抗的腹水效价可达１０７，抗原识别位点为淋球菌外膜蛋白约３５ｋＤ的糖蛋白成分．在免疫荧光实验中，２株单抗可与不同型淋球菌标准株反应，不与有关的细菌和真菌交叉。     

A study on the polymorphism of human MHC class I-related MR1 gene and identification of an MR1-like pseudogene

Human MR1 is a recently discovered, ubiquitously transcribed gene very similar to the HLA class I loci and of unknown function. Mouse and rat MR1 sequences have also been described showing high similarity with the human gene. The goal of this work was to investigate if human MR1 was polymorphic. We have found that DNA sequences of MR1-specific polymerase chain reaction (PCR) products obtained from samples of diverse ethnic origin were invariant except in one case in which two silent mutations were detected. We also found an MR1-like sequence displaying significant differences with the previously described, the most remarkable of which is a STOP codon in the alpha2 domain indicating that is a pseudogene.     

Mutations in gyrA,gyrB, parC,and parE in quinolone-resistant strains of Neisseria gonorrhoeae

Mutations in the genes for the subunits GyrA and ParC of the target enzymes DNA gyrase and topoisomerase IV are important mechanisms of resistance in quinolone-resistant bacteria, including Neisseria gonorrhoeae. The target enzymes also consist of the subunits GyrB and ParE, respectively, though their role in quinolone-resistance has not been fully investigated. We sequenced the quinolone-resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE in 25 ciprofloxacin-resistant strains from Bangladesh (MIC 4-->32 mg/l) and 5 susceptible strains of N. gonorrhoeae. All the resistant strains had three or four mutations. Two of these were at positions 91 and 95 of gyrA. Fourteen strains had an additional mutation in parC at position 91, and 17 strains had an additional mutation in parE in position 439. No alterations were found in gyrB. The five susceptible strains had identical DNA sequences. Data indicate that the mutations detected in the QRDR of gyrA and parC may be important in the development of quinolone resistance. According to transformation experiments we assume that the alteration in parE is not related to a high degree of quinolone resistance. There was no correlation between ciprofloxacin MICs and pattern or number of mutations in the target genes.     

Prevalence,phenotypic and genetic characteristics of prolyliminopeptidase‐negative Neisseria gonorrhoeae isolates in Sweden during 2000–2007

Johansson E, Fredlund H, Unemo M. Prevalence, phenotypic and genetic characteristics of prolyliminopeptidase‐negative Neisseria gonorrhoeae isolates in Sweden during 2000–2007. APMIS 2009; 117: 900–4. In Neisseria gonorrhoeae culture diagnostics, species confirmation is commonly performed using commercial biochemical tests relying on prolyliminopeptidase (PIP) activity. It was previously shown that one PIP‐negative strain was mainly globally transmitted during 2000–2004. The aims were to investigate the prevalence and phenotypic and genetic characteristics of PIP‐negative N. gonorrhoeae isolates in Sweden during 2000–2007. Gonococcal isolates (n = 1230) cultured in Sweden during 2000–2007 were characterized using PIP screening, antibiogram, serovar determination, pip and porB gene sequencing, and N. gonorrhoeae multiantigen sequence typing (NG‐MAST). Fifteen (1.2%) of the isolates were PIP‐negative. Of those, 13 (87%) were indistinguishable to the previously described globally transmitted strain, i.e. displayed serovar IB‐4 (Bpyvut), similar antibiograms, ST210 (n = 10)/ST292 (n = 3) and contained an identical single nucleotide pip gene deletion. Wherever high reliance is placed on PIP activity for N. gonorrhoeae species confirmation, changes in the diagnostic strategies may need to be considered and/or monitoring of the PIP activity is crucial.     

The Neisseria gonorrhoeae population in Sweden during 2005-phenotypes, genotypes and antibiotic resistance

In Sweden, the gonorrhoea incidence has significantly increased since an all-time low in 1996. We aimed to phenotypically and genotypically characterise N. gonorrhoeae isolates (n=180) transmitted in Sweden during 2005. All isolates were susceptible to cefixime, ceftriaxone, and spectinomycin. However, 2%, 50% and 75% displayed intermediate susceptibility or resistance to azithromycin, ciprofloxacin and ampicillin, respectively. The isolates were assigned to 28 different serovars using Genetic Systems monoclonal antibodies (Mabs) (discriminatory index, 91.0%) and 46 different serovars using Pharmacia Mabs (index, 94.4%). Furthermore, they displayed 95 porB sequences (index, 97.8%) and 95 N. gonorrhoeae multiantigen sequence typing (NG-MAST) sequence types (STs) (index, 98.0%). 51 (54%) of these STs have not been previously described. 14 ST clusters, comprising between 3 and 15 isolates, were identified that indicate the existence of several transmission chains. The high number of unique STs (n=63) may be associated with import of strains from abroad, local emergence of new STs, incomplete epidemiological surveillance, and/or suboptimal diagnostics, including contact tracing. Overall, the Swedish N. gonorrhoeae population was remarkably diversified. Comprehensive knowledge regarding transmission, phenotypes (including antibiotic resistance), but also in many cases highly discriminative and precise genotypic characteristics of the N. gonorrhoeae strains circulating in our societies, is crucial.     

Incidence, epidemiology and evolution of reduced susceptibility to ciprofloxacin in Neisseria gonorrhoeae in Korea

Objective: To verify the decrease of susceptibility to ciprofloxacin in Neisseria gonorrhoeae , determine the size of the recently reported new β-lactamase plasmid and explain the high prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG).
Methods: Gonococci were isolated from prostitutes in Korea. Antimicrobial susceptibility was tested by NCCLS disk diffusion and agar dilution methods. Plasmid was isolated by an alkaline lysis method. Patterns of Nhe l-digested genomic DNA were compared after pulsed-field gel electrophoresis (PFGE).
Results: The minimum inhibitory concentration of ciprofloxacin for 50% of the isolates rose from 0.015 mg/L in 1993 to 0.12 mg/L in 1996. The proportion of PPNG remained at 70% or over during the 5-year period. The size of a novel β-lactamase plasmid, first reported in 1994, was determined to be approximately 3.2 MDa, and 48% of the PPNG isolates contained it. Twelve of 50 isolates had the same PFGE pattern and nine others another pattern.
Conclusion: The rapid decrease of fluoroquinolone-susceptible gonococci suggests that in the near future the drug may become less useful for gonorrhea treatment. The new 3.2-MDa plasmid may have been introduced as a result of the recent increase in overseas travel. The PFGE pattern suggests that high prevalence of PPNG may be due to dissemination of a few resistant clones among the high-risk groups.     

STD门诊患者沙眼衣原体、解脲支原体、淋球菌与人乳头瘤病毒感染情况分析

目的 了解本地区STD门诊患者沙眼衣原体(CT)、解脲支原体(UU)、淋球菌(NG)与人乳头瘤病毒(HPV)感染情况,从而指导临床诊断与用药.方法 采用聚合酶链式反应(PCR)结合Taqman技术,对采集自来院就诊STD门诊患者的女性宫颈口分泌物、男性尿道口分泌物及相关尿液标本中的特异性DNA核酸片段进行荧光PCR检测.结果 CT、UU、NG、HPV四种病原体总感染率分别为7.49％、42.92％、3.11％和3.06％,除HPV外其它三种病原菌感染率存在性别差异;其中以UU感染率居首位,女性明显高于男性;二重感染率为5.76％,三重感染率为0.83％,未见四重感染,其中以CT和UU二重感染最常见;不同年龄段感染情况显示,以40岁以下年龄段感染率最高.结论 将HPV纳入泌尿生殖感染检测很重要;通过本研究也发现,无论单一病原体感染还是混合感染,除HPV外均存在明显的性别分布差异,而且两种感染的性别分布相一致;40岁以下年龄段为主要传染源和高危人群,应作为性传播疾病防治工作的重点;双重感染和三重感染较常见,应引起高度重视;目前未发现四重感染.     

不孕不育者生殖道淋球菌、沙眼衣原体和解脲支原体的检测结果分析

目的了解在不孕不育患者中生殖道淋球菌（NC）、沙眼衣原体（CT）、解脲支原体（UU）感染的情况。方法应用实时荧光定量PCR方法对465例不孕不育患者生殖道3种病原体基因进行定量测定。结果阳性检出268例，总阳性率为57．64％，其中UU的阳性检出率最高，占43．87％；重叠感染中以UU＋CT的阳性检出率最高，占3．44％。结论不孕不育患者生殖道中3种病原体感染率不尽相同，尤以UU感染率最高，是造成不孕不育的重要原因。     

Comparison of microscopy, culture and in-house PCR and NASBA assays for diagnosis of Neisseria gonorrhoeae in Russia

This study aimed to assess the laboratory diagnosis of Neisseria gonorrhoeae in St. Petersburg, Russia. In total, 334 consecutive symptomatic patients were enrolled. Cervical and urethral specimens from women (n=286) and urethral specimens from men (n=48) were analyzed by microscopy, culture and two in-house NAATs, i.e. polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA), developed in Russia. All N. gonorrhoeae-positive samples were confirmed using porA pseudogene and 16S rRNA gene sequencing. All methods displayed 100% specificity, i.e. positive predictive values of 100%. Compared to the PCR (most sensitive method in the present study), in women the sensitivity of both microscopy and culture was 31.8%, and that of NASBA was 90.9%. In men, microscopy, culture and NASBA displayed a sensitivity of 75%, 50% and 100%, respectively. The negative predictive values of microscopy, culture, and NASBA were 97.3%, 97.3%, and 99.6% in women, and 97.8%, 95.7%, and 100% in men, respectively. According to the PCR, the prevalences of N. gonorrhoeae were 4.5% (women) and 8.3% (men). In conclusion, both the investigated Russian NAATs displayed a high sensitivity and specificity. However, in general the diagnosis of gonorrhoea in Russia is suboptimal and crucially requires validation, improvements and quality assurance.     

Epidemilogical analysis of Neisseria gonorrhoeae isolates by antimicrobial susceptibility testing, auxotyping and serotyping

PURPOSE: This study was carried out to analyze the epidemiology of gonorrhea based on antimicrobial susceptibility testing, auxotyping and serotyping in New Delhi, India. METHODS: Sixty gonococcal isolates from males with urethritis, females with endocervicitis and their sexual contacts were studied. The isolates were subjected to antimicrobial susceptibility testing, auxotyping and serotyping for epidemiological characterization. RESULTS: We observed nine antibiotic resistance patterns. Ninety-eight percent of isolates were resistant to ciprofloxacin, while 20% isolates were penicillinase producing N. gonorrhoeae (PPNG) and 18.3% isolates were tetracycline resistant N. gonorrhoeae (TRNG). Eight auxotypes were observed, of which the NR (non-requiring), proline requiring and arginine requiring were most common auxotypes. On the basis of serotyping alone, the gonococcal isolates could be differentiated into three serogroups and 18 serovars. Serogroup WI represented 46.7% and WII/III represented 51.7% of isolates and one strain was WI and WII/WIII serogroup combination. When results of auxotyping and serotyping were combined (A/S) 29 A/S classes could be identified. The most prevalent A/S classes were NR/Aost, NR/Arost, Pro/Aost and Pro/Boprt. CONCLUSIONS: Although A/S typing had the highest discriminatory index, isolates recovered from index case and their sexual contacts were found to be identical by all typing methods.     

淋病奈瑟菌临床菌株外膜蛋白PIA基因序列分析及原核表达系统的构建

目的： 分析本地区淋病奈瑟菌临床菌株外膜蛋白PIA基因核苷酸及氨基酸序列，构建PIA基因的原核表达系统。方法：采用高保真PCR扩增9株淋病奈瑟菌全长PIA基因序列，T-A克隆测序后与GenBank公布的序列进行同源性比较。构建PIA原核表达系统。采用不同浓度IPTG诱导重组目的蛋白rPIA表达，10%SDS-PAGE和Bio-Rad凝胶图像分析系统检测rPIA表达情况。采用Ni-NTA亲和层析法提纯rPIA，SDS-PAGE检测提纯效果。结果： 与报道的PIA基因序列（GenBank No: L19962）比较，9株淋病奈瑟菌核苷酸和氨基酸序列相似性分别高达99.6%-100%和99.1%-100%，均属于IA6血清型。rPIA表达量可占细菌总蛋白量的50.1%，提纯后仅显示单一的目的蛋白条带。结论： IA6为本地区淋病奈瑟菌优势血清型，该基因序列相当保守。所构建的PIA基因原核表达系统能高效表达rPIA，为今后研制淋病奈瑟菌血清学检测试剂盒及疫苗研制奠定了基础。     

Antimicrobial susceptibility/resistance and molecular epidemiological characteristics of Neisseria gonorrhoeae in 2009 in Belarus

Increased antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global concern, and ultimately gonorrhoea may become untreatable. Nonetheless, AMR data from East-Europe are scarce beyond Russia, and no AMR data or other characteristics of gonococci have been reported from Belarus for more than 20 years. The aim was to describe the prevalence of AMR, and report molecular epidemiological characteristics of gonococci circulating in 2009 in Belarus. In a sample of 80 isolates, resistance prevalences to antimicrobials used for gonorrhoea treatment in Belarus were: Ceftriaxone 0%, spectinomycin 0%, azithromycin 17.3%, tetracycline 25.9%, ciprofloxacin 34.6% and erythromycin 59.2%. The isolates displayed no penA mosaic alleles, 38 porB gene sequences and 35 N. gonorrhoeae multiantigen sequence types, of which 20 have not been described before worldwide. Due to the high levels of antimicrobial resistance, only ceftriaxone and spectinomycin can be recommended for empirical treatment of gonorrhoea in Belarus according to WHO recommendations. Continuous gonococcal AMR surveillance in Eastern Europe is crucial. This is now initiated in Belarus using WHO protocols.     

淋病奈瑟菌rmp基因缺失突变株的构建及其抗体杀菌作用研究

目的研究淋病奈瑟菌的外膜蛋白Rmp在免疫阻抑中的作用及其消除策略。方法PCR扩增淋病奈瑟菌rmp基因,将rmp中间200个核苷酸残基用卡那霉素抗性基因Kan取代,含rmp两侧侧翼区和Kan的DNA片段△rmp∷Kan转化淋病奈瑟菌 WHO-A菌株,PCR和Western blot 鉴定野生rmp被突变基因（△rmp∷Kan）取代并不能表达Rmp的突变株。突变株免疫小鼠,并用抗体介导的补体杀菌作用研究免疫血清的抗菌活性。结果构建了rmp基因缺失的淋病奈瑟菌突变株,突变株诱生的抗体具有更强的杀伤淋病奈瑟菌活性。结论淋病奈瑟菌rmp基因缺失突变株可能消除了野生株中Rmp的免疫阻抑作用,在新型全细胞减毒活疫苗研制中具有潜在应用价值。     

叶酸代谢相关基因多态性及血浆同型半胱氨酸与唐氏综合征关系研究

目的研究叶酸代谢相关的亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MTH-FR)基因多态性与唐氏综合征(Down syndrome,DS)发生的关系。方法选择100例生育过DS患儿的汉族母亲及100名相匹配的正常对照组母亲,PCR-限制性片段长度多态性方法检测MTHFR677C/T的基因型,化学发光法检测血浆中同型半胱氨酸(homocysteine,HCY)的水平。结果病例组MTHFR677T等位基因的频率较对照组增高,差异有统计学意义(P=0.002);杂合基因型CT的比值比为2.12(95%CI:1.14～3.94);而纯合基因型TT的比值比为3.43(95%CI:1.41～8.36)。平均血浆HCY浓度在病例组[(9.04±3.85)μmol/L]较对照组[(6.53±2.06)μmol/L]增高,差异有统计学意义(P<0.01)。MTHFR677位点一个和(或)两个等位基因C→T的变异,不论在病例组还是对照组均可引起HCY水平的显著增加(P<0.01)。同为MTHFR677CC基因型,病例组中的血浆HCY浓度仍较对照组增高(P<0.01),这种增加不依赖于MTHFR的基因型。结论血浆HCY和叶酸代谢相关基因的遗传多态性是汉族妇女生育DS患儿的危险因素。     

Gonorrhoea surveillance, laboratory diagnosis and antimicrobial susceptibility testing of Neisseria gonorrhoeae in 11 countries of the eastern part of the WHO European region

Quality-assured worldwide surveillance of antimicrobial resistance (AMR) in Neisseria gonorrhoeae is crucial for public health purposes. In the countries of the eastern part of the WHO European region the knowledge regarding gonococcal AMR is limited, and antimicrobials of many different types, sources and quality are used for gonorrhoea treatment. This study surveyed gonorrhoea incidence, laboratory diagnosis and gonococcal AMR testing in 11 independent countries of the former Soviet Union. The national gonorrhoea incidences remain mainly high. In general, gonococcal culture and AMR testing were rarely performed, poorly standardized and rarely quality assured. To establish a gonococcal AMR surveillance programme in Eastern Europe, i.e. the geographical area of the former Soviet Union, several actions have recently been undertaken by the Eastern European Sexual and Reproductive Health (EE SRH) Network and the WHO. The information provided herein will be useful in this respect.     

Identifying a consensus sample type to test for Chlamydia trachomatis,Neisseria gonorrhoeae,Mycoplasma genitalium,Trichomonas vaginalis and human papillomavirus

ObjectivesSexually transmitted infections (STIs) are a global cause of acute illness. Early detection plays a crucial role in interrupting transmission and preventing complications. However, the accessibility of STI testing is curbed by the lack of an overall preferred sample type. By means of a prospective study in female sex workers (FSW), we compared the sensitivity of samples from different anatomical sites in detecting Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium and human papillomavirus. Besides, we documented the prevalence of each STI in this high-risk population.MethodsWe selected 303 FSW and tested them for each STI by nucleic acid amplification testing on two vaginal and cervical swabs from different manufacturers, cervical smear and first-void urine. The sensitivity of each sample type was compared for each infectious agent in order to identify a consensus sample type.ResultsVaginal swabs were superior to all other sample types, with an overall sensitivity of 86%. The sensitivity was the lowest for first-void urine, detecting only 63% of positive cases. The prevalence was 3.3% (10/299) for Neisseria gonorrhoeae; 9.0% (27/299) for Chlamydia trachomatis; 7.4% (22/298) for Trichomonas vaginalis; 10.8% (32/296) for Mycoplasma genitalium and 55.6% (158/284) for human papillomavirus.ConclusionsWhen testing for STIs, vaginal swabs are the sample of choice and first-void urine should be avoided. Designating (self-sampled) vaginal swabs as a consensus sample type enables harmonization of STI testing and extension of testing to large numbers of unscreened females.     

HER-2基因Ile655Val多态性与结直肠癌易感性分析

目的 探讨HER-2原癌基因Ile655Val多态性与结直肠癌易感性的相关性,及其在自然人群中的分布频率.方法 应用病例对照研究,对浙江省嘉善县292例结直肠癌患者和842名健康对照者采用聚合酶链反应-限制性片段长度多态性方法检测HER-2基因密码子655基因型.结果 结直肠癌组HER-2基因Ile/Val+Val/Val基因型频率(25.34%)和Val等位基因频率(13.36%)均显著高于对照组(18.41%和9.74%)(P<0.05).与Ile/lie基因型携带者相比,Ile/Val+Val/Val基因型携带者患结直肠癌的风险增加(OR=1.54,95%CI:1.11～2.14).HER-2基因多态性与吸烟、饮酒的交互作用OR值分别为1.43(95%CI:0.88～2.30)和1.29(95%CI:0.73～2.29).结论 HER-2基因Ile655Val多态性与结直肠癌易感性相关,但是这种多态性与吸烟、饮酒在结直肠癌发生中不存在交互作用.     

X染色体上五个多态位点筛选及其多态性分析

目的从X染色体上筛选多态性较强的多态位点,为X连锁遗传病的连锁分析和基因定位奠定基础。方法利用BCMSearchLauncher程序从相关基因组序列中筛选短串联重复序列。利用Primer3设计扩增短串联重复序列的引物,采用PCR扩增技术和聚丙烯酰胺凝胶电泳方法,对所筛选出的短串联重复序列进行多态性分析。结果从X染色体相关区域中筛选出8个短串联重复序列,多态性分析表明,其中的5个短串联重复序列具有多态性,χ2检验表明,各位点的基因型分布符合Hardy-Weinberg平衡定律(P>0.05),且具有较高的杂合度。结论从X染色体上筛选出5个多态位点,可用于X染色体基因的连锁分析和基因定位。     

The angiotensin-converting enzyme (ACE) genetic polymorphism: its relationship with plasma ACE level and myocardial infarction

The angiotensin-converting enzyme (ACE) is a key factor in the production of angiotensin II and in the degradation of bradykinin, two important peptides involved in vascular physiology. Plasma and cellular ACE levels in humans are influenced by an insertion (I)/deletion (D) polymorphism of the ACE gene, the ACE I/D polymorphism. The D allele has a frequency of approximately 0.53 in Caucasian populations and is codominantly associated with higher levels of ACE. We have studied this polymorphism in a large multicenter case-control study (the ECTIM study) and found that the D allele was associated with a parental history of fatal myocardial infarction (MI) in the controls and was more frequent in male patients with MI than in controls. This case-control difference was compatible with a codominant effect of allele D on the risk of MI with relative risks of 1.57 for DD vs II and 1.26 for ID vs II (test for trend p < 0.003). In subjects at low risk of MI (plasma ApoB < 1.25 g/1 and body mass index < 26 kg/m²), the relative risk of DD vs ID + II was 2.7 (p < 0.0005). The results were very homogeneous in the four populations included in the study. In a family study, using linkage-segregation analysis, we have shown that the ACE I/D polymorphism is a marker for an unknown functional polymorphism (ACE S/s) which appears to be a new independent risk factor for MI.