河西走廊地域幽门螺杆菌vacA和cagA基因型分布研究

[目的]探讨河西走廊胃癌高发区胃癌患者幽门螺杆菌(Helicobacter pylori,Hp)vacA和cagA基因型的分布情况,为当地Hp流行病学研究和疫苗研制提供参考。[方法]复苏和纯培养从河西走廊地域医院收集并分离到的89株Hp,抽提DNA,设计vacA信号序列s1a、s1b、s1c和cagA引物,PCR扩增vacA信号序列以及cagA基因并鉴定,分析Hp菌株中vacA和cagA基因型分布以及不同基因型与患者临床病理类型的关系。[结果]89 Hp菌株vacA基因均阳性,其中信号序列为s1a型者占66.29%,s1c型占33.71%,未见s1b型。vacA s1a基因型在慢性胃炎、消化性溃疡、胃癌各组中的构成比均高于s1c基因型。不同病理类型的疾病间,vacA s1a和s1c基因型构成均差异无统计学意义(P0.05)。cagA阳性率为97.75%,cagA+菌株在慢性胃炎、消化性溃疡、胃癌各组中的构成比均高于cagA-菌株。不同病理类型的疾病间,cagA+和cagA-菌株构成均差异无统计学意义(P0.05)。[结论]河西走廊地域Hp菌株大多数为致病性高的Ⅰ型菌株;vacA信号序列以s1a为主,其次为s1c;cagA+的高毒力菌株分布广泛,这可能是当地上消化道疾病高发的重要原因。     

幽门螺杆菌感染BALB/c小鼠模型的研究

目的　建立稳定的Hp感染BALB/c小鼠模型 ,用于Hp致病、免疫和防治的研究。 方法　选用临床分离株 ,应用外科手术法向小鼠胃内直接接种Hp菌液 0 2ml(10 9CFU/ml) ;接种后不同时间取小鼠胃粘膜组织进行细菌学和病理学检查。结果　接种后 2～ 8周 ,小鼠胃内均可分离到Hp ,Hp分离阳性率为 95 %。病理学检查显示鼠胃粘膜明显炎症。感染鼠用阿莫西林治疗后 ,鼠胃组织Hp培养均转阴性。结论　使用Hp鼠胃接种法 ,能建立稳定的Hp感染BALB/c小鼠模型     

乙型肝炎病毒感染BALB/C小鼠动物模型建立的实验研究

[目的]建立小鼠急性乙型肝炎病毒(HBV)感染的动物模型,观察小干扰RNA(si RNA)在体内对HBV复制和表达的影响.[方法]通过尾静脉快速、高负荷注射含1.5倍的HBV真核表达质粒(pHBV1.5).注射后第6天,MEIA检测血清HBsAg、HBeAg的水平;RT-PCR检测肝组织HBV prec/c mRNA转录的水平.同时设立pcDNA3.1正常对照组和PBS空白对照组.[结果]pHBV1.5注射组小鼠血清HBsAg、HBeAg为阳性,且肝组织表达HBeAg;而pcDNA3.1和PBS对照组未能检测到HBsAg、HBeAg的表达.[结论]成功地建立了急性HBV感染的BALB/C小鼠动物模型,在一定程度上克服了由于HBV宿主范围极窄给人们研究带来的极大限制.     

雷公藤多苷对溃疡性结肠炎小鼠结肠黏膜TLR4信号通路表达的影响

[目的]观察雷公藤多苷对右旋葡聚糖硫酸钠(DSS)诱导的小鼠溃疡性结肠炎模型结肠黏膜LPS/TLR4信号通路表达的影响,探讨其治疗溃疡性结肠炎的可能作用机制。[方法]BALB/c小鼠随机分为6组:模型组,低、中及高剂量中药雷公藤多苷灌胃组、阴性对照组、正常对照组。采用DSS复制BALB/c小鼠溃疡性结肠炎动物模型,雷公藤多苷灌胃给药21d后,采用荧光定量PCR、western blot法检测TLR4mRNA和蛋白的表达。[结果]3组中药灌胃组较阳性对照组TLR4表达水平显著降低(P0.01),但中、高剂量中药灌胃组、阴性对照组与正常组两两之间表达水平差异无统计学意义(P0.05)。[结论]雷公藤多苷能够抑制LPS/TLR4信号通路的表达,抑制UC小鼠的炎症反应,对UC发病起到一定得保护作用。     

幽门螺杆菌BALB/c小鼠适应性菌株的驯化及模型建立

目的:通过体内连续传代获得幽门螺杆菌(Hpylori)的BALB/c小鼠适应性定植菌株并建立稳定的感染模型.方法:以H pylori蒙古沙鼠适应株GS_(10)连续传代感染BALB/c小鼠,每次感染20只,感染后4 wk进行微生物学检查(分离培养、直接涂片染色镜检、快速尿素酶试验、PCR鉴定),观察H pylori的定植情况,计算每批动物的感染率,直至感染率稳定在80%以上,然后对已稳定感染的小鼠胃黏膜组织进行H pylori定量培养及病理学检查.结果:随着H pylori菌株GS_(10)在BALB/c小鼠内的连续传代,感染率逐渐升高.GS_(10)株初次感染率仅为11.1%,传代至第6代(BS_6株)以后,感染率稳定在80%以上;BALB/c小鼠适应株BS_8感染小鼠后4 wk在胃窦、胃体和胃底的定植密度(CFU/g组织)的常数对数均值分别为5.32±0.88,4.14±1.05和1.05±2.25,与SS1的定植密度相比,相差不显著(P>0.05).感染鼠病理学检查在胃窦黏膜上皮细胞间及固有层中发现大量炎症细胞浸润;在胃窦及幽门部上皮细胞表层黏液、胃腺窝中见到大量H pylori存在.结论:经过连续传代感染,驯化出一株高定植力和感染率的Hpylori BALB/c小鼠适应株,并成功建立稳定的H pylori小鼠感染模型.     

SjESG-2A锤头状核酶表达载体构建及其抗日本血吸虫虫卵发育研究

目的设计并合成日本血吸虫中国大陆株卵壳蛋白基因2A(eggshell protein gene 2A,ESG-2A)的特异性锤头状核酶并构建真核表达载体。探讨其在BALB/c小鼠体内的抗日本血吸虫虫卵发育作用。方法运用计算机软件人工设计并合成核酶基因(RZ),将核酶基因克隆到真核表达载体pcDNA3.1(+)中,采用双酶切、琼脂糖凝胶电泳及DNA序列测定方法鉴定阳性克隆。以日本血吸虫尾蚴感染BALB/c小鼠,建立动物模型。大量抽提阳性重组子,分别在小鼠感染时、感染后2w、感染后4w,将其经后腿胫前肌免疫BALB/c小鼠,共3次。小鼠感染6w后计数成虫负荷及肝组织虫卵数。ELISA法检测小鼠脾淋巴细胞培养上清IFN-γ和IL-4的水平。结果经酶切电泳及DNA测序证实合成的核酶基因序列正确并已被准确克隆入pcDNA3.1(+)的XbaI和EcoR I酶切位点之间,命名为pcRz-ESG-2A。在核酶组,IFN-γ的水平呈上升趋势,IL-4的水平呈先上升后下降;在空载体组和生理盐水组,IFN-γ的水平呈上升趋势,IL-4的水平也呈上升趋势。核酶组免疫BALB/c小鼠能诱生减虫率25.64%和减卵率44.21%。结论成功合成SjESG-2A特异性的锤头状核酶基因并构建了该基因的真核表达载体。核酶在已感染日本血吸虫尾蚴的BALB/c小鼠体内具有抗日本血吸虫虫卵发育的作用。     

经口感染弓形虫诱导小鼠黏膜免疫动物模型的建立

目的建立经口感染弓形虫速殖子诱导黏膜免疫的动物模型。方法BALB/c小鼠分别灌胃接种5×103、5×104、5×105、5×106个RH株速殖子,观察小鼠的体况、病理变化;检测肠道分泌型IgA(SIgA)和Peyer’spatches(PP)淋巴细胞及小肠上皮内淋巴细胞(IEL)T细胞亚群的变化。结果经口接种5×104个弓形虫RH株速殖子可使小鼠出现临床症状和病理改变;SIgA水平升高;黏膜诱导部位的CD4 T亚群及效应部位的CD8 T亚群水平升高。结论5×104个弓形虫RH株速殖子灌胃接种小鼠可以诱导机体黏膜免疫应答。     

弓形虫Chinese 1基因型Wh6弱毒株感染小鼠抗强毒株致死性感染的免疫保护作用

目的 研究我国流行的优势基因型Chinese 1弱毒Wh6株感染抗致死性强毒Wh3株攻击感染的保护力,探讨其潜在的制备减毒活疫苗的价值。方法 将30只BALB/c雌性小鼠随机分为3组(I,II,III组),每组10只。取弱毒株弓形虫保种小鼠脑组织,分离包囊,计数。I组小鼠灌胃包囊25-35个;II组给与PBS对照;III组正常小鼠不予任何干预。35 d后,I组感染小鼠脑组织压片镜检,查见典型弓形虫包囊,表明慢性弓形虫感染BALB/c小鼠模型建立成功。分别取Wh3株速殖子(3 000个),腹腔感染I组和II组小鼠,观察小鼠存活及腹腔虫荷;同时剖杀小鼠,收集脾细胞培养上清,采用流式细胞术、荧光定量PCR、酶联免疫吸附试验等检测脾细胞中的Th1、Th2细胞因子水平。结果 II组对照鼠在感染Wh3株弓形虫速殖子后第7 d内死亡5只,第8 d剩余5只全部死亡,随机取3只死亡小鼠对其腹腔进行计数,计数结果为(1.28±0.035 1)×10⁶/mL;而I组小鼠截止实验结束全部存活(P<0.01)。II组鼠脾细胞培养上清Th1细胞因子IFN-γ、IL-12和Th2细胞因子IL-10水平相比I组显著升高(P<0.001)。其中Th1细胞因子升高程度明显高于Th2细胞因子,巨噬细胞向Th1方向极化。结论 用弱毒株Wh6包囊攻击感染BALB/c鼠,可诱导强力的抵抗Wh3强毒株致死性感染的免疫保护力。Chinese 1基因型Wh6株具有候选减毒活虫疫苗研究的潜在价值。     

雷公藤多苷片对溃疡性结肠炎小鼠结肠巨噬细胞自噬的影响

[目的]观察雷公藤多苷片对右旋葡聚糖硫酸钠(DSS)诱导的小鼠溃疡性结肠炎(UC)模型结肠巨噬细胞自噬相关蛋白——自噬蛋白微管相关蛋白1轻链3(LC3)的表达变化,探讨其治疗UC的可能作用机制。[方法]SPF级雌性BALB/c小鼠随机分为4组:正常对照组、雷公藤多苷片混悬液灌胃组和美沙拉嗪片混悬液灌胃组和模型对照组。采用DSS复制BALB/c小鼠UC动物模型,随机分组后分别予美沙拉嗪片混悬液、雷公藤多苷片混悬液及蒸馏水灌胃给药28d后,取出结肠组织,分离培养结肠巨噬细胞,采用Western blot法检测自噬相关蛋白LC3II/I的表达。[结果]雷公藤多苷片混悬液灌胃组和美沙拉嗪片混悬液灌胃组较模型对照组自噬相关蛋白LC3II/I的表达明显下降。[结论]雷公藤多苷片能够通过抑制细胞自噬,抑制UC小鼠的炎症反应,对UC发病起到一定得保护作用。     

IFN-γ基因敲除小鼠的繁育及其对TgCatCHn4弓形虫的感染研究

目的 本研究目的是繁育和鉴别IFN-γ基因敲除小鼠,并探究IFN-γ基因敲除小鼠对TgCatCHn4弓形虫虫株的感染情况。方法 从美国Jackson 试验室购入IFN-γ基因敲除小鼠冷冻胚胎,由北京协和医院复苏。IFN-γ^+/-小鼠按雌雄3∶1合笼饲养繁殖,取小鼠尾尖组织,提取DNA,经PCR方法鉴定IFN-γ基因表达情况。IFN-γ^-/-小鼠和BALB/c小鼠经皮下注射TgCatCHn4弓形虫,分析小鼠的感染率及存活情况,镜检小鼠大脑包囊数量和和肺脏中的速殖子形态。弓形虫阳性小鼠组织接种于Vero细胞,观察弓形虫的生长情况。结果 IFN-γ^-/-小鼠的繁育和基因鉴定获得成功。感染TgCatCHn4弓形虫虫株后,IFN-γ^-/-小鼠存活时间17.0±1.0 DPI,肺脏涂片可见大量弓形虫速殖子。BALB/c小鼠存活时间304.0±43.7 DPI,包囊数量2.6±1.7个/只小鼠大脑。结论 获得IFN-γ^-/-小鼠,其感染弓形虫后存活时间短,是理想的急性弓形虫病的动物模型。     

CagA+幽门螺杆菌裸鼠感染模型的建立

目的用携带ｃａｇＡ基因的Ｈｐ成功感染ＢＡＬＢ／ｃ裸鼠。方法分别采用ｃａｇＡ基因阳性和阴性的具有较强动力的新鲜Ｈｐ分离菌株，通过灌胃方式感染ＢＡＬＢ／ｃ裸鼠，感染后４周和８周分两批处死，进行微生物学和病理组织学检查。结果灌喂８周后，ｃａｇＡ＋实验组和ｃａｇＡ－实验组均成功定植Ｈｐ（１６／１６）；ｃａｇＡ＋组胃粘膜表现明显炎症及坏死病变，而ｃａｇＡ－组仅有轻微炎症发生。结论建立典型的Ｈｐ动物感染模型应选择ｃａｇＡ＋菌株。     

Helicobacter pylori Cytotoxic Genotype Is Associated With Peptic Ulcer and Influences Serology

Objective : We studied 146 patients with peptic ulcer disease (  n = 72  ), antral gastritis (  n = 58  ), or duodenitis (  n = 16  ) to ascertain whether the cytotoxic genotype of Helicobacter pylori (Hp) is associated with peptic ulcer disease and/or antral gastritis and whether it influences the circulating levels of total anti-Hp antibodies, anti- cagA antibodies, and pepsinogens. Methods : A gastric juice sample was obtained from each patient. After DNA extraction, polymerase chain reaction was used to amplify the genes urease A ( ureA ), cagA , and vacA of Hp. Results : A significant association was found between peptic ulcer disease and the cytotoxic genotypes, characterized by the presence of s1 and m1 alleles of vacA and by cagA. Patients with a cagA -positive genotype showed a significant increase in anti- cagA antibodies and also had significantly increased circulating levels of pepsinogen C. Conclusions : Cytotoxic Hp strains are mainly involved in determining peptic ulcer disease, but not antral gastritis. The higher levels of circulating pepsinogen C found in patients infected with cytotoxic genotypes may reflect the higher degree of inflammation sustained by these strains.     

贵阳地区幽门螺杆菌临床分离株中ureA、cagA、vacA、iceA基因的分布及分析

目的了解贵阳地区临床分离的幽门螺杆菌（Hp）的毒力基因ureA、cagA、vacA、iceA的分布特征,探讨不同毒力基因型与上消化道疾病的关系。方法用特异的16SrDNA聚合酶链反应进行临床分离Hp的菌种鉴定,对经过鉴定的152株幽门螺杆菌进行ureA、cagA、vacA、iceA基因及亚型的PCR检测。结果 ureA基因的检出率为100%（152/152）,vacA基因的检出率为100%（152/152）,vacA基因亚型以s1a-m2型为主,占76.3%（116/152）,cagA基因检出率为39.5%（60/152）,ieeA1基因检出率36.8%（56/152）,iceA2基因检出率为34.2%（52/152）,13.2%（20/152）的菌株iceA1和iceA2基因均阳性,不同基因型菌株在慢性胃炎和消化性溃疡中的检出率无统计学意义（P〉0.05）。结论贵阳地区幽门螺杆菌毒力基因vacA以s1a-m2型为主,cagA阴性比例高于cagA阳性,不同基因型菌株与消化性疾病间无明显相关性。     

The importance of vacA, cagA, and iceA genotypes of Helicobacter pylori infection in peptic ulcer disease and gastroesophageal reflux disease

OBJECTIVE: To study the relationship between the presence of H. pylori virulence factors and clinical outcome in H. pylori infected patients. METHODS: DNA was isolated from an antral biopsy sample and vacA, cagA, and iceA genotype were determined by PCR and a reverse hybridization technique in 183 patients with culture-proven H. pylori infection: 51 with peptic ulcer disease (PUD), 62 with gastroesophageal reflux disease (GERD), and 70 with a normal endoscopy (gastritis only; GO). RESULTS: Forty-four samples (24%) showed more than one allelic variant in the vacA s- or in-region and/or both iceA1 and iceA2 genotypes, indicating multiple strain infection. These were excluded from statistical analysis. vacA s1 and cagA were significantly more common in PUD than in GERD and GO. Logistic regression analysis showed that GERD patients were more often infected with strains lacking both cagA and iceA than GO patients (OR = 0.36; CI = 0.15-0.89). Trend analysis showed that GERD patients were most often infected with less virulent strains (p < 0.002). CONCLUSION: Multiple strain infection is common. H. pylori strains possessing the vacA s1 genotype and/or cagA are associated with PUD. GERD patients, infected with H. pylori, mostly carry less virulent strains possessing neither cagA nor iceA1. Our findings support the hypothesis that virulent strains protect against the development of GERD.     

Helicobacter pylori cagA, vacA and iceA genotypes in northern Thai patients with gastric disease

Helicobacterpylori, a common infectious bacterium, has been linked to chronic gastritis, peptic ulcer and gastric cancer. Gastric biopsy specimens were obtained from 58 northern Thai patients with gastritis, 28 with gastric ulcer, 45 with duodenal ulcer and 4 with gastric cancer. cagA, vacA s1 and iceA gene was found in 88, 98, and 89% of the specimens, respectively. For vacA, the frequency of subtype s1a, s1c and combined sla and s1c was 40, 16, and 41%, respectively. The frequency of subtype s1a/m1 and s1a/s1c/m1 was 27 and 20%, respectively. Fifty-three patients (39%) were infected with multiple vacA genotypes but there was no association with clinical outcome. cagA positive and mixed vacA s1a and s1c strains were found in significantly more cases of duodenal ulcer than gastritis (p < 0.05). For iceA, subtype iceA1 reached a frequency of 60%, whereas subtype iceA2 was only 24%.     

Analysis of Helicobacter pylori antimicrobial susceptibility and virulence genes in gastric mucosal biopsies in the United Arab Emirates

AIM: To determine the virulence attributes (presence of cagA and vacA genes) of Helicobacter pylori , and presence of clarithromycin resistance genes in gastric mucosal biopsy samples obtained in the United Arab Emirates. METHODS: DNA was extracted from antral gastric biopsy samples from 91 dyspeptic patients. Real-time PCR and melting curve analysis were used to identify patients infected with H. pylori and to further identify strains containing the A(2142/43)G or the A(2142)C mutations that are associated with clarithromycin resistance. PCR was also used to identify cagA - and vacA -positive strains. RESULTS: Real-time PCR analysis detected the presence of H. pylori in 55 (60%) samples. Thirty-six pathogen-positive samples contained at least one of three point mutations associated with clarithromycin resistance. The vacA gene was present in 40 (72.7%) and cagA was present in 41 (74.5%) of the positive samples. Both genes were present in 36 (65%) of the positive samples. The presence of each clarithromycin-inducing mutation was largely independent of the others. Mutation at one position, A(2142/43)G, was strongly associated with the presence of both the vacA gene and the cagA gene. CONCLUSIONS: A high proportion of gastric mucosal biopsies obtained in the UAE is positive for genes associated with clarithromycin resistance. This may have implications for treatment of the infection.     

Heterogeneity in the Helicobacter pylori vacA and cagA genes: association with gastroduodenal disease in South Africa?

BACKGROUND: Helicobacter pylori infection is universally associated with gastritis, but only sometimes with clinically significant disease. Candidate virulence markers seem to be useful in identifying the pathogenic infections in some populations. AIMS: To investigate the association between putative virulence markers and disease in an African population. METHODS: Fifty nine H pylori strains isolated from dyspeptic patients (11 with peptic ulceration, eight with gastric adenocarcinoma, and 28 with no pathology other than gastritis) were studied for differences in the genes vacA and cagA. RESULTS: Forty seven (80%) of 59 strains had the vacA signal sequence genotype s1 (one s1a, 46 s1b) and 12 (20%) had subtype s2. vacA mid-region analysis revealed that 40 (68%) strains were vacA m1 and 19 (32%) were m2. All 14 strains from patients with peptic ulceration were vacA s1, in contrast to 23 (66%) of 35 strains from patients with gastritis alone (p<0.01). vacA s2 was found exclusively in patients with gastritis alone (p<0.01). All strains isolated from patients with gastric adenocarcinoma were s1b/m1 (p<0. 005 versus gastritis alone). cagA was detectable in 56 (95%) of 59 isolates. Strains from patients with peptic ulceration (12/13 versus 19/30 with gastritis alone, p=0.05) had the shortest fragment length in the 3' region of cagA, while 4/10 strains from patients with gastric cancer had the longest fragment length in this region (p<0. 02 versus gastritis alone). CONCLUSION: In this study, the vacA s1 genotype, and fragment length of the 3' region of cagA identified isolates associated with significant clinical disease. The vacA s1bm1 genotype seems to be strongly associated with gastric cancer.     

Helicobacter pylori virulence factors--one part of a big picture

CONTEXT: At least half the world's population is infected with Helicobacter pylori, although only 10-20% of carriers develop gastric diseases, ranging from ulcer to MALT-lymphoma and adenocarcinoma (MALT is mucosa-associated lymphoid tissue). The clinical outcome of H pylori infection is determined by a complex interaction of environmental influences and host and microbial virulence factors. H pylori genotypes carrying the babA2 gene, encoding a bacterial adhesin mediating interaction with gastric epithelial cells, have enhanced pathogenicity. Moreover, coexistence of babA2 with other bacterial virulence factors further worsens clinical outcomes. STARTING POINT: To further elucidate the clinical relevance of babA2-genopositive H pylori strains, Carlo-Frederico Zambon and colleagues analysed the association of babA2 genotypes with gastritis, gastroduodenal ulcer disease, or intestinal metaplasia in 167 infected Italian individuals. The coexistence of babA2 with other potentially disease-related H pylori genes, such as cagA, vacA, or oipA, correlated with clinical outcome. 36% of H pylori strains were babA2(-) genopositive, and abundance of babA2 was associated with the genomic presence of the other potential virulence-factor genes. H pylori strains carrying babA2, cagA, and the vacA genotype s1m1 were associated with the highest risk of developing intestinal metaplasia, whereas this condition was rarely (<10%) associated with strains with a cagA-, babA2-, vacA s2m2 genotype. Whilst the risk of developing more serious gastric lesions increased as the number of virulence factor genes accumulated in a given H pylori strain, there was no indication of any one specific bacterial gene-pattern being associated with a particular clinical disease. WHERE NEXT? Identifying the factors responsible for the enhanced pathogenicity of H pylori leading to development of life-threatening diseases in a subset of infected individuals is a mandatory task for the future. Identification of virulence-associated H pylori genes and investigation of their clinical relevance in large prospective studies will help to define such strains with increased pathogenicity. The value of H pylori genotypes as predictors of disease outcome is limited, because the pathogenic impact of bacterial virulence factors is greatly influenced by coexisting environmental and host factors.     

上海地区幽门螺杆菌cagA基因3’区和vacA基因的多态性分析及其临床意义

探讨上海地区人群中幽门螺杆菌（H．pylori）cagA基因3’区和vacA基因的多态性及其临床意义。方法：99株H．pylori菌株分离自17例慢性浅表性胃炎（CSG）、21例慢性萎缩性胃炎（CAG）、19例胃溃疡（GU）、23例十二指肠溃疡（DU）和19例胃癌（GC）患者。用聚合酶链反应（PCR）技术对H．pylori菌株的cagA基因3’区和vacA基因信号序列及中间区等位基因进行扩增和检测。结果：99株H．pylori菌株中84株（84．8％）cagA基因阳性,其3’区产物大小均约650bp,属A型。vacA基因信号序列仅检出sla型,见于从94．1％（16／17）的CSG、952％（20／21）的CAG、89．5％（17／19）的GU、87．00（20／23）的DU和89．5％（17／19）的GC患者中分离的菌株（P＝0．87）;中间区等位基因仅检出m2型,见于从70．6％（12／17）的CSG、71．4％（15／21）的CAG、63．20（12／19）的GU、73．9％（17／23）的DU和57．9％（11／19）的GC患者中分局的菌株（P＝0．72）。结论：上海地区人群中H．pylori菌株的cagA基因3’区相对保守;绝大多数vacA基因属sla／m2型。本研究结果不支持这些基因的多态性与H．pylori感染临床结局相关的观点。     

Direct determination of Helicobacter pylori vacA genotypes and cagA gene in gastric biopsies and relationship to gastrointestinal diseases

OBJECTIVE: Our aim was to detect Helicobacter pylori (H. pylori) from gastric biopsies of 248 patients using a novel, polymerase chain reaction (PCR)-based methodology, which simultaneously facilitates the determination of H. pylori vacA genotypes and cagA gene. METHODS: A simple methodology for sample preparation was established and PCR was performed with primer systems for the 16S rRNA, vacA, and cagA genes, thus circumventing the need to culture H. pylori and to extract DNA from biopsy samples. RESULTS: Infection with H. pylori was detected in 147 (59.3%) of 248 patients. The vacA signal sequence genotype s1 was present in 104 (81.3%) of 128 H. pylori-positive patients, and 24 (18.8%) patients had the genotype s2. The vacA middle region types m1 and m2 were detected in 46 (35.9%) and 79 (61.7%) patients, respectively. The combinations s1/m2 (43%) and s1/m1 (35.9%) were found more frequently than s2/m2 (18.8%). The cagA gene was detected in 75 (72.1%) of 104 H. pylori-positive biopsies with the vacA genotype s1. All 24 biopsies with the type s2 were cagA negative. Strains of the type vacA s1 were found in 97% of H. pylori-positive patients with peptic ulcer disease and were associated with the presence of the cagA gene, whereas 96% of the strains of the type vacA s2 were detected in patients who only had nonulcer dyspepsia. CONCLUSIONS: Using a novel PCR-based methodology, H. pylori 16S rRNA gene, vacA genotypes, and cagA gene can now be rapidly detected directly in gastric biopsies with high accuracy. These data demonstrate that infection with H. pylori strains of the vacA s1 genotype and the cagA gene are more likely to result in peptic ulcer disease. Determination of vacA genotypes and cagA gene may contribute to the potential clinical identification of patients at different levels of risk.