Detection of Helicobacter pylori DNA in feces and saliva by polymerase chain reaction: a review

The polymerase chain reaction (PCR), known for its high sensitivity and specificity, has been used for the detection of Helicobacter pylori DNA in bodily materials such as feces and saliva. Since fecal specimens contain PCR inhibitors, DNA before PCR amplification has been purified using various biochemical, immunological and physical pre-PCR steps. Several PCR protocols, differing from each other in the selection of genomic targets and primers, have produced varying degrees of specificity and sensitivity in detecting H. pylori DNA. PCR identified antimicrobial resistance of H. pylori in feces. It also detected virulence factor genes such as the cytotoxin-associated gene (cagA) and vacuolating cytotoxin gene (vacA) in feces and saliva. While the cagA gene was detected in 50-60% of fecal specimens, it was found in 25% of salivary specimens from patients. There was considerable variation in the detection rate of H. pylori DNA in salivary samples. The detection rate in saliva with the most effective primer pair was lower than that observed in feces, making saliva a less suitable specimen for the diagnosis of H. pylori infection. There is controversy regarding the permanent presence of H. pylori in saliva. Whether the salivary and gastric specimens of an individual harbor identical or different strains has not been resolved. PCR cannot distinguish between living and dead organisms. However, it can offer quick results on fecal and salivary specimens, which may contain fastidious and slow-growing H. pylori in low numbers.     

Detection of Helicobacter pylori in water by direct PCR

AIMS: This paper demonstrates a rapid, simple method for the detection of Helicobacter pylori in water that eliminates the need for recovery of cells or DNA extraction prior to PCR. METHODS AND RESULTS: Direct polymerase chain reaction (DPCR) with primers specific for H. pylori ureA (urease, subunit A) were used to detect H. pylori added to groundwater. DPCR also detected H. pylori in a naturally contaminated water sample. CONCLUSIONS: DPCR should provide an improved method to assess contamination of water by H. pylori. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple, rapid method for detection of H. pylori in water will provide an improved means to investigate the possible role of water as a disease vector.     

Effects of Prolonged Chlorine Exposures upon PCR Detection of Helicobacter pylori DNA

The effect of low doses of free chlorine on the detection of Helicobacter pylori (H. pylori) cells by qPCR in tap water was monitored. Detection of sequences targeted to the ureA gene from preparations containing 107 cells/ml decreased about 2-4 logs by days 9 and 14, respectively. When duplicate suspensions of the 107 cells/ml were exposed to higher levels of chlorine, 0.2-2.2 mg/l, by day 9 and 14 there were 5 and 6 log decreases, respectively, in the detection of ureA gene. H. pylori target sequences (within suspended, intact cells at densities of 102-103 cells /ml) were rendered undetectable by qPCR analysis after 17 h of continuous exposure to low chlorine levels common to treated drinking water distribution systems. The persistence of DNA sequences within treated distribution systems detectable by qPCR may be as brief as 17 h especially for bacteria such as H. pylori which are known to occur in very low numbers within treated distribution systems. This study suggests that degradation of H. pylori DNA target sequences by chlorine levels commonly found within treated water distribution systems occurs within the average water retention times (2-3 days) commonly found in these systems.     

原位PCR对胃癌和胃炎组织内幽门螺旋杆菌的检测

为探讨幽门螺旋杆菌(Helicobacter pylori, Hpy)与胃癌发病的关系,我们建立了原位(PCR(in situ PCR,ISPCR)方法,并使用它对胃炎和胃癌组织内的幽门螺旋杆菌进行了检测。发现在胃炎组织内Hpy的感染率为83.3％,胃癌组织内的感染率为65％。在11例胃癌阳性标本中,4例为细胞核阳性,2例细胞浆阳性,5例为所带正常胃腺组织内阳性。提示,Hpy的致癌机理,除与细菌的长期刺激外,尚可能与其参与细胞的功能调控有关。     

口腔中幽门螺杆菌PCR检测方法的建立

全世界大约有50%的人口感染幽门螺杆菌(Helicobacter pylori, Hp),Hp感染容易造成慢性胃炎、消化性溃疡、低度恶性胃MALT淋巴瘤及胃癌,对人类造成严重的危害。为寻求一种简便、准确、非创伤性的方法用于口腔中Hp的检测,本研究建立了口腔中幽门螺杆菌的PCR检测方法,并对建立的PCR方法的敏感性、特异性进行分析,并应用于检测唾液样品和牙菌斑共50份。结果显示,50份样品中,阳性率为82%。结果表明,本研究建立的口腔中Hp的PCR检测方法特异性强、敏感性高,可以用于临床上Hp诊断的重要参考。     

An alternative, sensitive method to detect Helicobacter pylori DNA in feces

Background: Despite the high sensitivity and specificity of PCR, detection of Helicobacter pylori DNA in feces is still challenging. Fecal samples contain inhibitory molecules that can prevent amplification of the target DNA. Even by using specific DNA extraction kits for stools, monitoring of infection by analyzing stool samples remains problematic and endorses the need for improved diagnostic methods. Materials and Methods: The newly proposed method uses selective hybridization of target DNA with biotin‐labeled probes, followed by DNA isolation with streptavidin‐coated magnetic beads. After three washing steps, the purified DNA can be amplified immediately using conventional or quantitative PCR. In order to test this technique on biological samples, Mongolian gerbils were infected with H. pylori ATCC 43504 and fecal samples were analyzed on days 1, 4, and 10 post infection. Results: A detection limit of one bacterial cell per 100 mg stool sample was established, but only after removal of the magnetic beads from the target DNA by heating. This resulted in a 10‐fold increase of sensitivity compared to a commercially available stool DNA extraction kit. Analysis of fecal samples from infected gerbils demonstrated the presence of H. pylori DNA on each time point, while the uninfected animal remained negative. Conclusions: The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.     

Efficient removal of PCR inhibitors using agarose-embedded DNA preparations.

The use of agarose blocks containing embedded DNA improves the PCR amplification from templates naturally contaminated with polysaccharides or humic acids, two powerful PCR inhibitors. Presumably, the difference in size between the DNA macromolecules and these contaminants allows their effective removal from the agarose blocks by diffusion during the washing steps, whereas genomic DNA remains trapped within them. In addition, agarose-embedded DNA can be directly used for PCR since low melting point agarose does not interfere with the reaction. This simple and inexpensive method is also convenient for genomic DNAs extracted by other procedures, and it is potentially useful for samples containing other kinds of soluble inhibitors, overcoming this important problem of current amplification techniques.     

Detection of kinetoplast DNA of Trypanosoma cruzi from dried feces of triatomine bugs by PCR.

It is important to clarify the distribution of infected triatomine bugs in the endemic area of Chagas' disease for proper control. In the present study, we tried to detect T. cruzi kinetoplast DNA by PCR from dried triatomine feces collected from the house wall of an endemic area to assess the distribution of infected bugs more easily. The primers (P35/P36) were chosen to amplify the conserved region within the minirepeats of T. cruzi kinetoplast minicircle DNA. The kinetoplast DNA of T. cruzi could be actually detected in the dried feces collected from the wall of a brick-built house in Santa Cruz, Bolivia. Next, we examined the stability of T. cruzi kinetoplast DNA in the feces exposed to artificial environments. T. cruzi DNA was also detected by PCR in the feces left for 26 weeks at 25 degrees C and in those left for 4 weeks at 40 degrees C. The present study indicates that examination of dried feces on the wall can be an effective tool for surveillance of the natural infection of triatomine bugs that live in houses.     

Detection of Helicobacter pylori in saliva and esophagus

The route of Helicobacter pylori transmission remains unclear and the currently suggested route is person-to-person transfer by faecal-oral and oral-oral mode. The aim of this study was to verify the presence of H. pylori in esophagus and saliva of humans. Saliva samples, mucosal biopsies from esophagus, gastric antrum and fundus were collected from 19 patients with positive Urea Breath Test (UBT). Gastric biopsies were used for H. pylori colture and antimicrobial susceptibility tests whereas saliva samples were collected to detect H. pylori with a Nested-PCR targeting 16S rRNA gene as well as esophagus biopsies which were also investigated with immunohistochemical staining. Helicobacter pylori was isolated in 18 patients both in gastric antrum and fundus. The molecular analysis, confirmed by comparative sequences evaluation, gave positive results in all saliva and esophageal samples whereas the immunohistochemistry revealed the presence of H. pylori in 15.8% (3/19) of the esophagus samples. Our data suggest that saliva and esophagus may be considered reservoirs for H. pylori in humans and emphasize the need to use more susceptible techniques for H. pylori detection, in particular in over-crowded sites. Identification of the transmission route of H. pylori is crucial in developing an effective plan of surveillance by finding new means of disease management.     

Detection of Helicobacter pylori in cow's milk

AIMS: To investigate the existence of Helicobacter pylori in cow's milk as one of the foods which most Japanese children eat. METHODS AND RESULTS: Detection of H. pylori was demonstrated by the semi-nested polymerase chain reaction (PCR), a culture method and electron microscopy. Semi-nested PCR demonstrated the ureA gene of H. pylori in 13 of 18 (72.2%) raw milk samples and in 11 of 20 (55%) commercial pasteurized milk samples. Helicobacter pylori binding immunomagnetic beads with H. pylori-specific goat anti-H. pylori antibody was shown by electron microscopy in both raw and pasteurized milk positive for the ureA gene. Helicobacter pylori was cultured in one raw milk sample, whereas it was not cultured in pasteurized milk samples. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: There is a possibility that cow's milk is a transmission vehicle in childhood H. pylori infection, although we failed to confirm the survival of H. pylori in pasteurized milk.     

Detection of Helicobacter pylori DNA in drinking water biofilms: implications for transmission in early life

AIMS: To provide evidence of water quality as a risk factor for acquisition of Helicobacter pylori in early life, and to identify evidence for its presence within pots used to store drinking water. METHODS AND RESULTS: A prospective cohort study of 65 infants was conducted in the rural village of Keneba, The Gambia. Age of H. pylori colonization was determined and water pot biofilms were tested for H. pylori by sequencing of amplified DNA. Use of supplemental water was a strong risk factor for H. pylori colonization in infants (OR 4.71, 95% CI 1.17-22.5). DNA with 95% homology to the 16S rRNA gene of H. pylori was isolated from biofilms of water pots. CONCLUSIONS: Drinking water may be a reservoir for H. pylori in areas of the developing world where water quality is poor. Early introduction of water, particularly if stored in, or collected from contaminated sources, may be associated with an increased rate of H. pylori colonization.     

PCR detection of Helicobacter pylori in string-absorbed gastric juice

Molecular methods for detection of Helicobacter pylori infection have been shown to be highly sensitive in gastric biopsies and cultures. The objective of this work was to compare PCR detection of H. pylori DNA in string-absorbed gastric juice and in gastric biopsies. The study was performed in 47 dyspeptic adult patients undergoing endoscopy, and infection was detected by amplification of a segment of H. pylori ureA gene. Of the 29 patients positive in biopsy analysis, 23 (79%) were also positive in the gastric string. PCR analysis of gastric strings is a sensitive and safe procedure to detect H. pylori when endoscopy is not indicated, and may be of great clinical and epidemiological usefulness in determining effectiveness of eradication therapies, typing virulence genes and detecting antibiotic resistance mutations.     

Determination of Helicobacter pylori vacA allelic types by single-step multiplex PCR

AIMS: To develop and evaluate a novel multiplex PCR assay that enables definition of Helicobacter pylori vacA allelic type in a single reaction. METHODS AND RESULTS: Application of the one-step system to DNA extracts from 22 cultures of known vacA genotype demonstrated that it was highly accurate. Analysis of 15 matched gastric biopsy/culture pairs generated exactly correlating genotype profiles. vacA genotypes were determined from an additional 62/70 gastric biopsies from dyspeptic patients of known H. pylori positive status by the one-step assay, compared with 63/70 by the original two-reaction test. Types s1/m1, s1/m2 and s2/m2 were identified in 51.9%, 31.2% and 16.9% of biopsies, respectively. CONCLUSIONS: The multiplex PCR system developed enables rapid one-step vacA genotyping that is accurate, easy to interpret and more economical than the alternative multiple-reaction tests. Application of this system to gastric biopsies from patients in South-east England demonstrated that s1/m1 was the most common genotype, while s1/m2 and s2/m2 were less prevalent. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple one-step system can be applied direct to antral gastric biopsies without the need for culture, thereby facilitating rapid surveillance of vacA genotype in relation to geographical location and disease status.     

Detection of Helicobacter pylori in sewage and water using a new quantitative PCR method with SYBR green

Aims: To demonstrate the application of a new quantitative polymerase chain reaction (qPCR) technique for the determination of Helicobacter pylori concentrations in water, and to use this method to investigate the occurrence of the bacteria in sewage. The other aim was to study the survival capacity and detectability of the bacteria in artificially contaminated groundwater at different temperatures of 4 and 15°C. Methods and Results: The detection of H. pylori in water was aided by PCR using specific primers designed for the amplification of a fragment within the major vacuolating cytotoxin gene. Conventional culture was compared with conventional PCR and the new real-time (RT) qPCR approach for the quantification of the bacterium. Helicobacter pylori remained culturable for 120 h at 4°C as opposed to only 24 h at 15°C. RT qPCR demonstrated a 100-fold greater sensitivity for the detection of H. pylori DNA in comparison with conventional PCR. Scanning electron microscopic (SEM) observation showed that the normal spiral form changed to a coccoid form after 24 and 72 h at 15 and 4°C, respectively. Helicobacter pylori was found at 2–28 cells ml⁻¹ in sewage, of the 23 sewage samples – 84% were positive for H. pylori species-specific vacuolating cyctotoxin gene (vacA) by RT qPCR, but were negative by conventional PCR. Conclusions: The RT qPCR assay provided a specific, sensitive and rapid method for the quantitative detection of H. pylori in sewage. This molecular method would be valuable in studying the prevalence of H. pylori as required by the United States Environmental Protection Agency Contaminant Candidate List, particularly in nondisinfected ground waters, in sewage as a source of contamination, and for addressing the possible presence of viable but nonculturable of H. pylori. Significance and Impact of the Study: The quantitative detection of H. pylori by rapid and less-expensive methods than the TaqMan Assay using SYBR green could be an important tool to monitor infection in community by measuring the concentrations in sewage and to meet the new regulatory and risk-based frameworks for water supplies.     

Detection of free and plankton-associated Helicobacter pylori in seawater

AIMS: To detect both free and plankton-associated Helicobacter pylori in seawater samples collected on the Italian coast of the Adriatic Sea using a nested-PCR. METHODS AND RESULTS: Dissolved oxygen, pH, salinity and chlorophyll 'a' were the parameters recorded together with the characterization of zooplanktonic organisms. Plankton-associated H. pylori DNA was searched for in water samples filtered through 200 and 64 microm nylon nets whereas free bacteria were retained with the subsequent filtration through 0.22 microm pore-size membranes. Nested-PCR using primers for the glmM (ureC) gene was performed to reveal the presence of H. pylori. The DNA sequencing of amplified products confirmed the specificity of the assay. The sensitivity of the nested-PCR assay for H. pylori detection was 62 CFU per 100 ml in spiked water samples. Helicobacter pylori either free or bound to planktonic organisms was found in seven of 12 monthly samples. In particular, free bacteria were detected during the summer sampling and in November, December and March associated to planktonic cells. CONCLUSIONS: The presence of free and plankton-associated H. pylori in seawater suggests that it can be a significant reservoir and a potential route of transmission for the microorganism. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study seems to provide a promising background to define new and effective strategies for surveillance of this human pathogen.     

应用real-timePCR法快速定量人类粪便中双歧杆菌的研究

目的建立快速、准确从粪便标本中定量双歧杆菌的RT—PCR技术。方法传统培养定量法,普通PCR定量法,real—timePCR比较测量。结果（I）粪便标本前处理采取简单的离心和清洗、稀释步骤能去除粪便标本中的抑制物,实现不提取DNA直接进行PCR、real—time定量粪便中双歧杆菌。（2）本实验建立的PCR方法直接半定量粪便双歧杆菌技术在双歧杆菌值介于10^3～10^7CFU／ml时具有较好的分辨率,粪便标本普通PCR得理论菌数与培养得菌数值之间差异无显著性（P〉0．05）;real-timePCR直接定量双歧杆菌技术在双歧杆菌值介于10^1-10^7CFU／ml时具有较好的分辨率,粪便标本RT—PCR得理论菌数与培养得菌数值之间差异无显著性（P〉0．05）。结论利用PCR、real—timePCR直接半定量和定量粪便中的双歧杆菌可行。     

Repair and antirepair DNA helicases in Helicobacter pylori

Orthologs of RecG and RuvABC are highly conserved among prokaryotes; in Escherichia coli, they participate in independent pathways that branch migrate Holliday junctions during recombinational DNA repair. RecG also has been shown to directly convert stalled replication forks into Holliday junctions. The bacterium Helicobacter pylori, with remarkably high levels of recombination, possesses RecG and RuvABC homologs, but in contrast to E. coli, H. pylori RecG limits recombinational repair. We now show that the RuvABC pathway plays the prominent, if not exclusive, repair role. By introducing an E. coli resolvase (RusA) into H. pylori, the repair and recombination phenotypes of the ruvB mutant but not the recG mutant were improved. Our results indicate that RecG and RuvB compete for Holliday junction structures in recombinational repair, but since a classic RecG resolvase is absent from H. pylori, deployment of the RecG pathway is lethal. We propose that evolutionary loss of the H. pylori RecG resolvase provides an "antirepair" pathway allowing for selection of varied strains. Such competition between repair and antirepair provides a novel mechanism to maximize fitness at a bacterial population level.     

Detection of Helicobacter pylori in bile of cats

Lymphocytic cholangitis (LC) in cats is a biliary disease of unknown etiology. Helicobacter spp. were recently implicated in human primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC). Because of the similarities between PSC/PBC with LC, we hypothesized that Helicobacter spp. are involved in feline LC. A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from feline bile samples. Four of the 15 (26%) LC samples were positive, whereas only 8/51 (16%) of non-LC samples were PCR positive (p=0.44). Sequence analysis of the amplicons revealed a 100% identity with the Helicobacter pylori specific DNA fragments. Our data suggest an etiological role of H. pylori in feline LC and that cats are a potential zoonotic reservoir.     

Detection of Helicobacter pylori by PCR but not culture in water and biofilm samples from drinking water distribution systems in England

AIMS: To investigate treated water distribution systems in England as a source of Helicobacter pylori. METHODS AND RESULTS: Water and biofilms were obtained from 11 domestic and seven educational properties and from hydrants, reservoirs and water meters supplied by three water utilities. Samples were cultured on nonselective and antibiotic containing media combined with immunomagnetic separation concentration. Viable helicobacters were not detected in any of the 151 samples but Helicobacter-specific PCR assays detected DNA in 26% of samples from domestic properties, schools and hydrants with the highest frequency in biofilms (42%). Direct sequencing of six selected amplicons confirmed >95% sequence homology to H. pylori. CONCLUSIONS: While viable helicobacters were not isolated, evidence was obtained for the presence of Helicobacter DNA, including that of H. pylori. Biofilms on surfaces within water distribution systems may act either as sites for the passive accumulation of helicobacters or as potentially important reservoirs of infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings strengthen evidence that H. pylori may be transmitted through drinking water. However, there is currently no evidence that viable cells can survive the disinfection levels used in UK mains supplies and the health risk from this source remains unclear.