Helicobacter pylori test and medical reimbursement

There are an estimated 60 million people with Helicobacter pylori(H. pylori) infection who occupied 50% of the population of Japan. In Japanese medical reimbursement H. pylori tests were introduced on November 1, 2000 and they are able to use only to patients with gastric and duodenal ulcer. H. pylori tests were composed of rapid urease test, urea breath test, antibody test, bacterial culture and pathologic test. Payment of each test is 700 Yen. Classification and cost of H. pylori tests are shown. Usage of laboratory tests for H. pylori infection is mentioned. Those particular tests are useful to decrease the number of gastric and duodenal ulcer in Japan.     

Correlation of serum antibody titres with invasive methods for rapid detection of Helicobacter pylori infections in symptomatic children

Helicobacter pylori (H. pylori) is causally associated with peptic ulcer disease and gastric carcinoma. Typically, children get infected during the first decade of life, but diseases associated with H. pylori are seen mainly in adults. Multiple diagnostic methods are available for the detection of H. pylori infection. The aim of this study was to evaluate the correlation and diagnostic accuracy of three invasive methods [rapid urease test (RUT), histology and bacterial culture] and one non-invasive method (IgG serology) for diagnosis of H. pylori infection in a prospective cohort study conducted on 50 symptomatic children between two and eighteen years of age. Endoscopies with gastric biopsies were performed for RUT, culture and histopathological examination, respectively. IgG antibodies were measured in patient sera using a commercially available enzyme-linked immunosorbent assay (ELISA). RUT and positive H. pylori IgG antibodies were concordant in 88% (44/50) of patients. Both tests were negative in 32% (16/50), and both were positive in 56% (28/50). Disagreement occurred in 12% (6/50) of the patients: three of them (6%) had positive RUT and negative H. pylori IgG, and another three (6%) had negative RUT and positive H. pylori IgG. A combination of RUT with non-invasive serology constituted the optimum approach to the diagnosis of H. pylori infection in symptomatic children. The non-invasive serological test (ELISA) could not be used alone as the gold standard because it cannot distinguish between active and recently treated infection; and bacterial culture could not be used alone because of its low sensitivity.     

Evaluation of CLO test and polymerase chain reaction for biopsy-dependent diagnosis of Helicobacter pylori infection

Helicobacter pylori is now recognized as possibly playing an etiologic role on the development of chronic gastritis, peptic ulcers and adenocarcinoma of the distal stomach. CLO test and polymerase chain reaction (PCR) assay are rapid, biopsy-dependent diagnostic tests for H. pylori identification. In this study, we assessed four diagnostic methods (CLO test, PCR assay, culture and histological examination) for H. pylori detection in biopsy specimens from 78 patients with gastroduodenal diseases and investigating the efficiency of CLO test and PCR assay for the diagnosis of H. pylori infection. H. pylori was identified in 75.6%, 75.6%, 64.1%, 69.2% of patients by CLO test, PCR assay, culture and histological examination, respectively. Compared with the detection of H. pylori by culture and/or histological examination, the sensitivity and specificity of the CLO test were 98.2% and 81.8%, respectively, whereas the sensitivity and specificity of PCR assay were 96.4% and 77.3%, respectively. According to the H. pylori infection state as determined from the results of three concordant tests, the sensitivities of culture, CLO test, histological examination, and PCR assay were 90.9%, 96.4%, 98.2% and 100%, respectively. Whereas, the specificity was 100%, 95%, 95% and 90% for culture, CLO test, histological examination, and PCR assay, respectively. We found that both CLO test and PCR assay were highly sensitive and specific for H. pylori identification; however, PCR assay was more sensitive than other methods for detecting the specimens after patients received treatment. The results of this study suggest that CLO test is a rapid and sensitive method of screening for H. pylori infection and that PCR assay could provide an accurate indication of the state of infection both during treatment for eradication of H. pylori and at follow-up.     

Helicobacter pylori.

Helicobacter pylori is a gram-negative bacterium which causes chronic gastritis and plays important roles in peptic ulcer disease, gastric carcinoma, and gastric lymphoma. H. pylori has been found in the stomachs of humans in all parts of the world. In developing countries, 70 to 90% of the population carries H. pylori. In developed countries, the prevalence of infection is lower. There appears to be no substantial reservoir of H. pylori aside from the human stomach. Transmission can occur by iatrogenic, fecal-oral, and oral-oral routes. H. pylori is able to colonize and persist in a unique biological niche within the gastric lumen. All fresh isolates of H. pylori express significant urease activity, which appears essential to the survival and pathogenesis of the bacterium. A variety of tests to diagnose H. pylori infection are now available. Histological examination of gastric tissue, culture, rapid urease testing, DNA probes, and PCR analysis, when used to test gastric tissue, all require endoscopy. In contrast, breath tests, serology, gastric juice PCR, and urinary excretion of [15N]ammonia are noninvasive tests that do not require endoscopy. In this review, we highlight advances in the detection of the presence of the organism and methods of differentiating among types of H. pylori, and we provide a background for appropriate chemotherapy of the infection.     

Diagnosis of Helicobacter pylori infections

Hardly any other bacterial infection can be diagnosed with a similar variety of non-invasive and invasive tests as infection with H. pylori. Efficacy and specificity of well-proven tests such as urease test in biopsy specimens, histology, culture, and 13C breath test have been uniformly evidenced in numerous studies. Novel tests include molecular microbiological procedures, providing new opportunities for rapid detection of virulence factors and resistance genes, as well as antigen detection in feces. Though some open questions still need to be clarified, the latter test will gain major importance in the future. With the availability of the breath test and antigen detection in stool the need for the use of serological tests has been reduced, all the more so as the majority of the available quick tests have been shown to be of inadequate reliability. Well defined indications for the treatment of H. pylori infections are a major prerequisite for a reasonable use of this diagnostic armory.     

Microbiology--laboratory examinations for bacterias

As it has been required to identify pathogenic microbes in shorter times, simple and rapid methods have been developed and used. Here, we summarized the present situation of rapid diagnostic testing in clinical microbiology in Japan, and also presented our results on PBP2' detection. The rapid test kits available in Japan for E. coli, Helicobacter pylori, Salmonella, Streptococcus and Staphylococcus aureus were described. Rapid examination methods are based mainly on immunologic reactions, which included slide agglutination using latex particle, immunochromatography and ELISA. Times required for the identification are 10 to 15 minutes. Moreover, rapid test kits employing PCR are also marketed. Further, we evaluated MRSA-LA "Seiken" which is a rapid detection kit for PBP2' produced by MRSA. The test was shown to be highly sensitive and specific. For the rapid identification of pathogenic microbes, simple and rapid test kits described here will be used more in clinical diagnosis.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> and <Emphasis Type="Italic">cagA</Emphasis> and <Emphasis Type="Italic">vacA</Emphasis> gene status in children from Brazil with chronic gastritis

Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1-16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

Real-time quantitative PCR for detection of Helicobacter pylori

Helicobacter pylori is one of the most common chronic infections in humans, in whom it is a key etiological factor in peptic ulcer disease, gastric mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. Humans are the bacterium's only host. Here we report the development of a real-time quantitative (Q) PCR-based assay to measure ureC gene copy number to detect H. pylori, based on the fact that there is only one copy of the ureC gene per bacterium. Upon optimization of LightCycler Q-PCR conditions, we obtained a standard curve with a linear range (correlation coefficient = 1) across six logs of DNA concentration. We were able to accurately quantify as few as 1,000 bacteria in our assay. Analysis of variance on 15 randomly selected clinical samples showed good reproducibility of this assay. Comparison of Q-PCR results with bacterial culture and histopathological results from an additional 85 clinical biopsy samples showed a significant difference for the presence of H. pylori. Many samples that were negative for H. pylori by culture and histopathology were positive by Q-PCR. Contamination of PCR by H. pylori or H. pylori genetic material could not be ruled out. In summary, we developed a rapid, sensitive, and real-time Q-PCR method for detecting H. pylori. This technique offers a significant improvement over other available methods for detecting H. pylori in clinical and research samples.     

Reliable detection of macrolide-resistant Helicobacter pylori via fluorescence in situ hybridization in formalin-fixed tissue.

Macrolide-resistant Helicobacter (H.) pylori represent an increasing therapeutic problem. Macrolide resistance is usually determined phenotypically in vitro with methods such as E-test or agar dilution test. A prerequisite for those tests, however, is bacterial culture that is not routinely set up in the course of gastroscopy. In contrast, formalin-fixed, paraffin-embedded biopsies are regularly available from patients who have undergone gastroscopy. In such biopsies macrolide-resistant H. pylori can be detected by the genotype-based technique of fluorescence in situ hybridization (FISH). Experience gained by this new method, however, is still extremely limited, especially in formalin-fixed tissue. Therefore, we retrospectively investigated formalin-fixed, paraffin-embedded biopsy specimens by FISH in 104 patients suffering from therapy-resistant H. pylori gastritis. To test the accuracy of FISH, we initially examined specimens from 53 patients for whom results of the E-test were available. Next we analyzed biopsies from another 51 patients that had been selected since phenotypical resistance testing had failed despite documented culturing attempts. In all 104 patients, H. pylori was detected by FISH and could thus be investigated for macrolide resistance. Overall, macrolide-resistant bacteria were found in 71 patients (68.3%). In 49 of 53 patients (92.4%), FISH and E-test returned identical results (no significant discordance according to McNemar's chi(2)-test). Taken together, our study demonstrates that FISH is a highly sensitive and reliable method for detecting macrolide-resistant H. pylori in formalin-fixed, paraffin-embedded biopsy specimens, which represents the routine method of processing tissue obtained upon gastroscopy.     

Which test is best for Helicobacter pylori? A cost-effectiveness model using decision analysis

GPs face a potential dilemma in deciding which test to use for detection of Helicobacter pylori. For patients with dyspepsia, the National Institute for Health and Clinical Excellence (NICE) advises primary care practitioners to adopt a 'test and treat' policy before considering a referral for gastroscopy. There are many ways of testing: serology, urea breath test, and faecal antigen test. NICE does not advocate any preferred single test for detecting H. pylori. In the current study a multi-stakeholder 2-day workshop was established to agree and populate a cost-effectiveness decision analysis model. The aim was to analyse the three types of tests available for H. pylori and to determine which is the most practical and cost effective. Agreement on the costs and diagnostic values to be entered into the decision-analytic model was achieved. Results indicate that the faecal antigen test was the most effective in terms of true outcomes and cost. One thousand virtual patients were allocated to each of the three tests. Serology had 903, urea breath test had 961, and the faecal antigen test had 968 true positive outcomes. Data indicate that the faecal antigen test is the preferable strategy for diagnosis of H. pylori in primary care. This has implications for implementing new testing processes and for commissioning new diagnostic pathways for use in primary care.     

Evaluation of diagnostic methods for Helicobacter pylori gastritis

The authors evaluated the use of direct Gram-stained smears, 1- and 24-hour urease broth tests, histologic examination, and culture to detect Helicobacter pylori in 100 gastric biopsy specimens from 97 patients with epigastric symptoms. Twenty-six patients had positive cultures and 27 had H. pylori identifiable in hematoxylin and eosin-stained sections. The gastric biopsy specimens from the 29 patients with culture and/or histologic findings positive for H. pylori showed active gastritis in 27 cases (93%), compared with 26 cases (37%) without H. pylori (P less than 0.0001). Chronic gastritis was present in 25 cases (86%) with H. pylori and 40 cases (56%) without H. pylori (P less than 0.01). Twenty patients had positive Gram-stained smears. Fifteen patients had positive 1-hour urease tests, and 3 had delayed positive 24-hour urease tests. There were no false-positive Gram's stain results, three false-positive 24-hour urease tests, two false-negative histologic results, and three false-negative cultures (one inadvertently incubated anaerobically). The sensitivities of the methods were as follows: 62% for the 24-hour urease test, 69% for direct Gram's stain, 90% for culture, and 93% for histologic examination. The authors conclude that the urease test used in this study has low sensitivity and limited specificity; that the direct Gram-stained smear is a useful, highly specific, rapid screening test; and that the lengthier methods of culture and histologic examination have comparably high sensitivity for the definitive diagnosis of H. pylori gastritis.     

Use of a combination of brushing technique and the loop-mediated isothermal amplification method as a novel, rapid, and safe system for detection of Helicobacter pylori

Gastric mucosal biopsy is widely used in the detection of Helicobacter pylori but is associated with a number of problems, including false-negative results due to sampling error and massive bleeding after biopsy. Given the extended period required to culture H. pylori, detection would be further improved by the use of rapid detection methods such as PCR. Here, we developed a rapid, safe, and convenient method for collecting H. pylori which combines endoscopic brushing with the loop-mediated isothermal amplification (LAMP) method. The specificity and sensitivity of LAMP were examined using nine urease-generating non-H. pylori bacterial species, Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Helicobacter hepaticus, and 51 H. pylori strains. Results showed that H. pylori-specific LAMP primers amplified H. pylori DNA only and that the lowest detection limit of the LAMP reaction was 10(2) CFU. Brushing and biopsy samples taken from 200 patients with peptic ulcer at Nagoya University Hospital and a regional health care center were subjected to both LAMP and culturing. No adverse effects such as severe bleeding or penetration occurred during the procedure. By LAMP assay, 123 patients were confirmed as H. pylori positive when brushing technique samples were assayed, whereas only 100 were positive when biopsy samples were assayed. Culture assay detected H. pylori in 117 patients when it was combined with the brushing technique and in 96 when it was combined with biopsy. Combination of the endoscopic brushing technique with LAMP is considered a useful and safe system for identifying H. pylori infection.     

Evaluation of a rapid urease test to detect Campylobacter pylori infection.

Seventy-five consecutive patients referred for upper gastrointestinal tract endoscopy were evaluated for Campylobacter pylori infection by pathology, culture, and a biochemical test to detect bacterial urease. Forty-eight patients (64%) had C. pylori present based on pathology or culture or both. Thirty-two urease tests were positive after 1 h, all in patients with C. pylori detected by the two other methods (specificity, 100%; sensitivity, 67%). After 24 h, 47 urease tests were positive, but only 40 had C. pylori present (specificity, 74%; sensitivity, 83%). When read after 1 h, the urease test was highly specific and led to rapid presumptive diagnosis.     

The efficacy of laboratory diagnosis of Helicobacter pylori infections in gastric biopsy specimens is related to bacterial density and vacA, cagA, and iceA genotypes

A total of 500 consecutive patients undergoing upper endoscopy were biopsied and tested for H. pylori infection by the Campylobacter-like organism (CLO) test, culture, histology, and PCR. Serum samples were tested by two different serological assays. Patients were considered H. pylori positive if at least two of the four biopsy specimen-based methods yielded positive results. PCR had the highest diagnostic sensitivity (99.4%), followed by histology (92.2%), culture (89.5%), and the CLO test (89.0%). The specificities of all methods were higher than 98%. Of the organisms from the 181 PCR-positive patients, the vacA (s and m regions), cagA, and iceA genotypes were determined by reverse hybridization (line probe assay) or an allele-specific PCR. Organisms that were detected by PCR but that remained undetected by the CLO test were significantly more often vacA s1 (P = 0.006), m1 (P = 0.028), and cagA positive (P = 0.029) than vacA s2, m2, and cagA negative, respectively. Organisms that were detected by PCR but that remained undetected by culture or histology more often contained iceA1 (P = 0. 034 and P = 0.029, respectively) than iceA2. Higher H. pylori density was associated with vacA s2 (P = 0.024), vacA m2 (P = 0.050), and cagA-negative (P = 0.035) genotypes. Also, the diagnostic results of the CLO test (P = 0.001) and culture (P = 0.031) but not those of the PCR (P = 0.130) were significantly associated with the H. pylori density. The rate of detection by the four biopsy specimen-based tests was lower for patients who used proton pump inhibitors, but this was independent of the H. pylori genotypes. These observations may be explained by different bacterial densities, as established by the distinct genotypes of H. pylori, and confirm that the biologies of strains with such genotypes are considerably different.     

Identification of Helicobacter pylori by immunological dot blot method based on reaction of a species-specific monoclonal antibody with a surface-exposed protein.

Monoclonal antibodies (MAbs) against membrane preparations of Helicobacter pylori were produced. One MAb was found to be specific for H. pylori, because it did not react with a number of other bacterial species, including Helicobacter felis and Campylobacter jejuni. This MAb reacted with a 30-kDa protein found in outer membrane preparations of H. pylori. The protein was also detected on the cell surface on intact bacteria when analyzed by immunoelectron microscopy. To facilitate the identification of H. pylori isolates after culturing of biopsies, an immunodot blot assay based on the reaction of this MAb was developed. This assay was found to be highly specific for H. pylori. Sixty-six clinical isolates typed as H. pylori by conventional biochemical tests were found to be positive, whereas no other bacterial species tested gave a positive result. By this method, reliable and rapid identification of H. pylori could be accomplished.     

Differentiation of metronidazole-sensitive and -resistant clinical isolates of Helicobacter pylori by immunoblotting with antisera to the RdxA protein

Antimicrobial resistance in Helicobacter pylori is a serious and increasing problem, and the development of rapid, reliable methods for detecting resistance would greatly improve the selection of antibiotics used to treat gastric infection with this organism. We assessed whether detection of the RdxA protein could provide the basis for determining the susceptibility of H. pylori to metronidazole. In order to raise polyclonal antisera to RdxA, we cloned the rdxA gene from H. pylori strain 26695 into the commercial expression vector pMAL-c2, purified the resultant fusion protein by affinity chromatography, and used this recombinant RdxA preparation to immunize rabbits. We then used this specific anti-RdxA antibody to perform immunoblotting on whole bacterial cell lysates of 17 metronidazole-sensitive and 27 metronidazole-resistant clinical isolates of H. pylori. While a 24-kDa immunoreactive band corresponding to the RdxA protein was observed in all metronidazole-sensitive strains, this band was absent in 25 of 27 resistant isolates. Our results indicate that testing for the absence of the RdxA protein would identify the majority of clinical isolates that will respond poorly to metronidazole-containing eradication regimens and have implications for the development of assays capable of detecting metronidazole resistance in H. pylori.     

Helicobacter pylori. Pathology and diagnostic strategies

Helicobacter pylori represents one of the most common and medically prominent infections worldwide. Infection with this microaerobic, gram-negative bacterium has been established as an etiologic factor in the development of peptic ulcer disease. In addition, H pylori infection has been associated firmly with the development of gastric neoplasia, including gastric adenocarcinomas and gastric mucosa-associated lymphoid tissue lymphomas. Effective antimicrobial treatment depends on sensitive and accurate diagnostic approaches. This review article discusses invasive and noninvasive strategies for diagnosis of H pylori infection. Invasive methods requiring endoscopic evaluation include bacteriologic culture and susceptibility testing, histopathologic studies, molecular diagnostics, and rapid urease testing. Noninvasive approaches include fecal antigen detection, serologic testing, and urea breath testing.     

Quantitative study of Helicobacter pylori in gastric mucus by competitive PCR using synthetic DNA fragments.

Helicobacter pylori is closely related to upper gastrointestinal diseases, and the precise evaluation of H. pylori infection is necessary for the treatment of these diseases. The aim of the present study was to establish a method for the quantitative detection of H. pylori. We applied a competitive PCR method using various amounts of synthetic DNA fragments containing the same primer-binding and a subset of the same template sequences as the target competing for primer binding and amplification in order to quantify H. pylori in gastric mucus. The results obtained by this method were compared with the results of histological examination, the rapid urease test, bacterial culture, the [13C]urea breath test, and urea and ammonia measurements in gastric juice. As the quantity of H. pylori in gastric mucus increased, the rates of positivity of histological examination, the rapid urease test, and bacterial culture increased. The quantity of H. pylori in gastric mucus was also significantly correlated with the results of the [13C]urea breath test and was negatively correlated with the urea/ammonia ratio in gastric juice. The competitive PCR method provides an objective measure of the quantity of H. pylori and makes it possible to distinguish true negatives from false negatives due to incomplete PCR and true positives from false positives due to contamination. This method is very useful for the precise evaluation of gastric H. pylori infection.     

Diagnosis of Helicobacter pylori infection in adults and children by using the Malakit Helicobacter pylori, a commercially available enzyme-linked immunosorbent assay.

Malakit Helicobacter pylori (Biolab, Limal, Belgium) is a second-generation enzyme-linked immunosorbent assay (ELISA) for the detection of Helicobacter pylori infection. We evaluated its ability to diagnose H. pylori infection in 489 asymptomatic pregnant women, 427 asymptomatic children, and 95 symptomatic children. 87 asymptomatic adults (17.8%), 31 asymptomatic children (7.3%), and 27 symptomatic children (28.4%) were seropositive. We observed an increase in H. pylori infection with age. 13C-urea breath tests were performed for all seropositive and 100 randomly selected seronegative asymptomatic adults. They were also performed for all seropositive and 65 randomly chosen seronegative asymptomatic children. Breath tests were positive for 86 of 87 (98.9%) seropositive adults, 30 of 31 seropositive children (96.8%), and no seronegative individual. Compared with those of culture, the sensitivity and specificity of the Malakit Helicobacter pylori were both 96%. We conclude that the Malakit Helicobacter pylori is equally suitable for adults and children. Therefore, this ELISA can be proposed as an important alternative to other more time-consuming and/or more expensive diagnostic tests for the detection of H. pylori.     

Use of serum-specific immunoglobulins A and G for detection of Helicobacter pylori infection in patients with chronic gastritis by immunoblot analysis.

Multiple invasive and noninvasive tests for detecting Helicobacter pylori infection are available. The current "gold standard" for the diagnosis of H. pylori infection requires histology and the rapid urease test. Our aim was to test the performance of immunoglobulin A (IgA) and IgG immunoblot assays in comparison with that of the gold standard tests for the diagnosis of H. pylori infection. Ninety patients who underwent gastroscopy were analyzed in a prospective study. Fifty-nine of them were defined to be H. pylori positive by the gold standard tests. The IgA and IgG immunoblot assays correctly identified H. pylori infection in 17 and 58 of these patients, respectively, indicating that determination of IgA antibodies seems to be of low diagnostic value for H. pylori infection. In contrast, the sensitivity and specificity of the IgG immunoblot assay were 98 and 71%, respectively, with positive and negative predictive values of 87 and 96%, respectively. Therefore, the IgG immunoblot assay proved to be a sensitive and useful, noninvasive test for the diagnosis of H. pylori infection.