A simple multiplex PCR assay for diagnosing virulent Helicobacter pylori infection in human gastric biopsy specimens from subjects with gastric carcinoma and other gastro-duodenal diseases

AIM: To evaluate and develop a multiplex polymerase chain reaction (PCR) assay for diagnosing and specific identification of virulent Helicobacter pylori strains and their main virulence genes cagA, cagE, cagT, vacA and hrgA. METHODS AND RESULTS: Genomic DNA from 82 gastric tissues was screened. A master pool of all the ingredients of multiplex reaction was prepared for amplification. Amplicons were sequenced to confirm the amplification of each target genes. Multiplex PCR assay was able to detect all the five target genes in 81.7% and deletions in one or more loci among 18.3%. Genotype cagT +ve/hrgA +ve/cagA +ve/cagE +ve/vacAs1 +ve was more predominant in this study population (67.07%). hrgA, cagT, cagE and cagA genes were present in 100%, 92.7%, 85.4% and 81.7% of the subjects, respectively. The vacAs1 subtype had higher prevalence frequency in patients with overt gastrointestinal disease (78.57%) than with GERD (gastro-esophageal reflux disease) and NUD (non-ulcer dispepsia) (50%). CONCLUSIONS: The multiplex PCR assay developed herein was able to genotype H. pylori isolates based on the main virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify H. pylori and the majority of their virulence gene markers by multiplex PCR assay represents a considerable advancement over other PCR-based methods for genotyping H. pylori from large population, and can be explored to gain insights at the genotypic variability exhibited by this pathogen.     

Development of a PCR-based technique for detection of Helicobacter pylori

Abstract A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori , using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.     

Helicobacter pylori stimulates a mixed adaptive immune response with a strong T-regulatory component in human gastric mucosa

BACKGROUND: Host factors play an important role in the pathophysiology of Helicobacter pylori infection and development of gastritis and related disease. The established opinion is that the T-cell-mediated immune response to H. pylori infection is of Th1 type. Our earlier immune cell phenotype studies indicate a mixed Th1-Th2 profile of the effector cells. Therefore, an extensive adaptive and regulatory cytokine gene expression profile was conducted by quantitative real-time polymerase chain reaction (qPCR). MATERIALS AND METHODS: Biopsies from gastric mucosa of 91 patients diagnosed as H. pylori negative, H. pylori positive with gastritis, or H. pylori positive with peptic ulcer were obtained by endoscopy. Gene expressions of nine cytokines and CagA status were measured by qPCR. RESULTS: All cytokine genes showed higher expression levels in the presence of H. pylori when compared to H. pylori-negative samples (fold increase: IL8: x 11.2; IL12A: x 2.4; TNF-alpha: x 5.2; IFN-gamma: x 4.3; IL4: x 3.6; IL6: x 14.7; and IL10: x 6.7). Patients infected with CagA-positive strains had higher expression of IL1-beta and IL18 compared to patients infected with CagA-negative strains (x 1.6 for IL1-beta and x 2.0 for IL18). Patients with duodenal ulcer had a lower antral Th1/Th2 ratio than other H. pylori-positive patients. CONCLUSIONS: The cytokine profile of H. pylori-infected gastric mucosa shows a mixed Th1-Th2 profile. Furthermore, a high IL10 expression may indicate that also regulatory T cells play a role in the chronic phase of H. pylori infection.     

Co-detection of Helicobacter pylori and of its resistance to clarithromycin by PCR

Our aim was to develop a rapid molecular test based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and making it possible to detect Helicobacter pylori directly from gastric biopsy samples, and to test its susceptibility to clarithromycin. A 629-bp fragment of the 23S rRNA gene of H. pylori was amplified by PCR and the mutations responsible for clarithromycin resistance were detected with BsaI and BbsI restriction endonucleases. Thirty-five gastric samples were tested in parallel by standard microbiologic methods (culture and clarithromycin susceptibility testing with E-test strips) and by PCR-RFLP. The 10 culture-negative samples were also PCR-negative. Sixteen out of the 25 culture-positive samples (64%) were PCR-positive. RFLP analysis could be done in 12 cases and the results were in agreement with those of the E-test: susceptibility in five cases, resistance in seven (six A2144G mutations and one A2143G mutation).     

Application of real-time PCR stool assay for Helicobacter pylori detection and clarithromycin susceptibility testing in Brazilian children

Background: Helicobacter pylori ClariRes assay is a novel commercially available real‐time PCR assay allowing H. pylori detection and clarithromycin susceptibility testing in either gastric biopsy or stool specimens. Objective: The aim of this study was to validate the novel biprobe real‐time assay in stool specimens from 217 dyspeptic children. Methods: DNA from gastric biopsies and stool specimens were obtained and submitted to the biprobe real time assay for H. pylori detection and clarithromycin susceptibility testing. Results: The sensitivity, specificity, and test accuracy were 69, 100 and 93.9% for the detection of H. pylori infection and 83.3, 100 and 95.6%, for detection of clarithromycin resistance. Conclusion: This assay proved to be appropriate for H. pylori clarithromycin susceptibility testing, particularly in children populations where a high prevalence of clarithromycin‐resistant strains is suspected.     

A real‐time PCR assay for detection and quantification of Lactococcus garvieae

Aims: To develop a rapid, sensitive, specific tool for the detection and quantification of Lactococcus garvieae in food and environmental samples. Methods and Results: A real‐time quantitative PCR (qPCR) assay with primers for CAU12F and CAU12R based on the 16S rRNA gene of L. garvieae was successfully established. The limit of detection for L. garvieae genomic DNA was 1 ng DNA in conventional PCR and 32 fg with a mean C_T value of 36·75 in qPCR. Quantification of L. garvieae vegetative cells was linear (R² = 0·99) over a 7‐log‐unit dynamic range down to ten L. garvieae cells. Conclusions: This method is highly specific, sensitive and reproducible for the detection of L. garvieae compared to gel‐based conventional PCR assays, thus providing precise quantification of L. garvieae in food and natural environments. Significance and Impact of the Study: This work provides efficient diagnostic and monitoring tools for the rapid identification of L. garvieae, an emerging pathogen in aquaculture and an occasional human pathogen from other members of the genus Lactobacillus.     

Multiplex PCR using 16S rRNA gene-targeted primers for the identification of bifidobacteria from human origin

Three multiplex polymerase chain reactions (PCRs) targeted on Bifidobacterium and related species were designed to identify human species. The selected primers yielded amplified products of various sizes, each specific for a species. Three to four pairs were gathered in one PCR reaction and their specificity under multiplex conditions was confirmed using DNA from 26 reference strains. Using this technique on unidentified faecal strains, B. bifidum, B. longum and B. breve species were commonly recovered in infants while B. adolescentis, B. catenulatum/B. pseudocatenulatum continuum and B. longum species were predominant in adults. Thus, a single PCR can provide the assignment of a strain to one these species, reducing the number of PCR reactions and hands-on time for the identification of human isolates of bifidobacteria. Moreover, this technique is also applicable for the in situ detection of bifidobacteria in DNA extracts from human stools.     

Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic tool

The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian-cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian-cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western-cagA positive and 53.7% (22/41) were East Asian-cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection.     

Development of PCR assays for detection of Streptococcus canis

Streptococcus canis isolates, also including S. canis of artificially contaminated milk, could be identified by polymerase chain reaction (PCR) amplification using oligonucleotide primers designed according to species-specific parts of the 16S rRNA gene and, after sequencing, according to S. canis-specific parts of the 16S-23S rDNA intergenic spacer region and with oligonucleotide primers detecting an internal fragment of the group G streptococcal CAMP factor gene cfg. The 16S rRNA gene- and CAMP factor gene cfg-specific oligonucleotide primers could be used together in a multiplex PCR. No cross-reactivities could be observed with other group G streptococcal isolates or with any of the other control strains of various streptococcal species and serogroups. The PCR methods presented in this study allowed a rapid and reliable identification of S. canis and might help to improve the diagnosis of this bacterial species in animal and human infections.     

A group-specific PCR assay for the detection of Helicobacteraceae in human gut

Background. Enterohepatic Helicobacter species are emerging pathogens, which are increasingly isolated from humans with enteric diseases. Nevertheless, current methods to detect Helicobacteraceae in the human gut have significant limitations. Methods. Based on 16S‐rRNA gene alignments and computer aided primer analysis a set of group‐specific PCR primers was designed. The evaluation of the PCR assay was performed using 36 ATCC reference strains and intestinal biopsies from 10 patients with defined gastric Helicobacter pylori status. The amplification products derived from clinical samples were cloned and subsequently analyzed by DNA sequencing. Sensitivity of the PCR‐assay was determined by spiking previously negative tested samples with decreasing amounts of Helicobacter DNA. Results. The analysis of the ATCC reference strains revealed amplification products in all 14 Helicobacter strains and Wolinella succinogenes, 21 other microorganisms representing negative controls did not produce PCR fragments. Four out of the 10 patient‐derived samples were positive. Three of them represented H. pylori‐derived DNA confirming the gastric H. pylori infection in these patients. In the fourth patient, who was suffering from Crohn's disease, H. pullorum was identified. The sensitivity of the PCR assay was 0.1 pg of Helicobacter‐derived DNA representing about 40 bacteria. Conclusion. The novel PCR assay described here is an important new tool in rapid and sensitive assessment for the presence of Helicobacteraceae in human gut.     

Polymerase chain reaction detection of bacterial 16S rRNA gene in human blood

Bacterial 16S ribosomal RNA genes (rDNA) were detected in blood samples from two healthy individuals by PCR under conditions involving 30 cycles that did not produce any visible products from negative control saline. Even from control samples, PCR involving 35-40 cycles yielded visible bands. Major clones detected in the blood samples, but not in control, were the Aquabacterium subgroup, Stenotrophomonas subgroup, Budvicia subgroup, Serratia subgroup, Bacillus subgroup and Flavobacteria subgroup. No clone was located within the bacteroides-clostridium-lactobacillus cluster, which is indigenous to gastrointestinal flora.     

Infection with cagA- and vacA-positive and -negative strains of Helicobacter pylori in a mouse model

To study the role of cytotoxin-associated protein (cagA) and vacuolating cytotoxin (vacA) in Helicobacter pylori infection in an experimental murine model, mice were infected with seven strains with different cagA and vacA status. Groups of 10 NMRI mice were challenged and were killed 5 weeks later. In a second study, 20 mice were challenged with a mixture of the same seven strains and killed 1, 3, 15 and 17 weeks post-inoculation. All seven strains were found to colonize the mice for the 5-week experimental period. Animals infected with vacA-positive strains, regardless of cagA status, showed an elevation of antibody titers. Two cagA-negative and vacA-positive strains and one cagA- and vacA-positive strain were found to 'take over' in the mixed infection as analyzed by the randomly amplified polymorphic DNA-polymerase chain reaction technique and in one mouse stomach we found coexistence of two of the strains. We found no evidence of the different strains colonizing different parts of the stomach.     

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.     

Following the course of human leptospirosis: evidence of a critical threshold for the vital prognosis using a quantitative PCR assay

In order to follow the course of acute human leptospirosis, an ELISA microtiter plate hybridization method was developed for the quantitative determination of Leptospira spp. in biological samples after PCR. The biotin-labelled amplified product (331 bp from the rrs gene) was hybridized with a complementary capture probe covalently linked onto aminated polystyrene wells, and detected using a colorimetric reaction. The mean detection limit was 50 copies per 10 microl. In a prospective study of human leptospirosis cases, we obtained evidence that a density of 10(4) leptospires per ml of blood is a critical threshold for the vital prognosis of the patients. The practicability of the method makes it suitable for use in tropical areas for multicentric studies. Such studies could lead to a better knowledge of the natural history of the human disease. The method is also suitable for experimental evaluation of improved antibiotic treatments for leptospirosis.