Serodiagnosis ofHelicobacter pylori infections by detection of immunoglobulin G antibodies using an immunoblot technique and enzyme immunoassay

A transferable solid phase enzyme immunoassay (TSP-EIA) and an immunoblot technique were evaluated for the detection of IgG antibodies againstHelicobacter pylori. Using the biopsy urease test as reference method, the sensitivity and specificity of the EIA were 96 % and 100 %, respectively. Immunoblot analysis was carried out by testing sera from patients with a positive urease test who suffered from type B gastritis, gastric and duodenal ulcers, and a negative control group. The immunoblottedHelicobacter pylori proteins showed reproducible immunoreactive bands at molecular weights of 130,93,75 and 67 kDa. The molecular weight protein fractions ofHelicobacter pylori of 180 kDa and higher were found to be of minor immunological significance. Proteins of less than 60 kDa exhibited wide serum-specific variations in reactivity after immunostaining. No correlation between specific immunoblot patterns and clinical signs induced byHelicobacter pylori infection was observed.     

Comparison of four stains and a urease test for rapid detection ofHelicobacter pylori in gastric biopsies

Gastric biopsies were obtained from 125 subjects to compare detection ofHelicobacter pylori by culture, a rapid urease test and histopathologic examination using haematoxylin-eosin, Gram, Giemsa, Warthin-Starry silver and acridine orange stains.Helicobacter pylori was isolated from 39 specimens. Acridine orange and Giemsa were the most sensitive stains, detecting 85 % and 79 % of positive specimens respectively. All stains showed high specificity (97–100 %). The sensitivity and specificity of the rapid urease test was 62 % and 100 % respectively. These stains or the rapid urease test may be useful for rapid detection ofHelicobacter pylori in gastric biopsies.     

Immune Response to Natural and Recombinant Antigens of Helicobacter pylori in Patients with Dyspeptic Complaints

 Sera of 223 dyspeptic patients with endoscopic findings of nonulcer dyspepsia (72%), gastric ulcer (15%) and duodenal ulcer (13%) were tested for antibodies against Helicobacter pylori with an enzyme immunoassay and an immunoblot technique using lysates of Helicobacter pylori cells as antigen source. One hundred and fifty-one (68%) sera were found to be positive for Helicobacter pylori IgG with both methods; 5% of the positive results in the enzyme immunoassay were false-positive due to cross-reactions mainly of proteins with a molecular mass of 43–66 kDa. Since cross-reactivity not only reduces the diagnostic value of the immunoassay but also complicates evaluation of the immunoblot results, an attempt was made to overcome these problems by using specific purified recombinant proteins instead of the crude cell preparations as antigens. Of the commonly recognised immunogens of Helicobacter pylori, antibodies against a cell surface protein of 26 kDa, the small urease subunit (29 kDa) and the cytotoxin-associated protein (130 kDa) were identified as highly sensitive serological markers for inclusion in a recombinant antigen mixture for Helicobacter pylori screening. Only the cytotoxinassociated protein was confirmed to be an indicator immunogen for ulcerogenic strains. To assess the reliability of recombinant fragments of this protein in serological screening, the reactivity of antibody to purified fragments of the cytotoxin-associated protein was compared with that to the natural protein. A C-terminal recombinant fragment of 58 kDa showed results identical to those obtained with the natural protein and was thus considered to be an appropriate component of an antigen mixture for serological detection of Helicobacter pylori.     

Presence ofHelicobacter pylori in the oral cavity,oesophagus, stomach and faeces of patients with gastritis

The presence ofHelicobacter pylori in the oral cavity (6 sites), oesophagus, stomach and bowel of 20 dyspeptic patients was investigated. Samples were cultured on three selective media and analyzed by 16S rDNA polymerase chain reaction (PCR) and southern hybridization.Helicobacter pylori DNA was detected by PCR from oral-cavity samples of three (20 %) and from faeces samples of only one (7 %) of the patients whose stomach biopsies were positive forHelicobacter pylori. When culture was used, the microorganism's rate of recovery from the oral cavity and faeces was 13 % and 7%, respectively. One patient had aHelicobacter pylori-like organism in samples collected from the tongue and palate. Both strains were urease, catalase and oxidase positive and grew microaerophilically but were negative on PCR analysis. This demonstrates the possibility of false identification ofHelicobacter pylori by use of routine enzyme reactions. Interestingly, specimens collected from the cheeks of three patients were positive forHelicobacter pylori by PCR analysis. This is the first instance of detection of this micro-organism in the cheek.     

Evaluation of a monoclonal antibody for detection ofHelicobacter pylori in a direct immunofluorescence test

A monoclonal antibody was developed for detection ofHelicobacter pylori in gastric tissue sections in a direct immunofluorescence test. On a comparison of the immunofluorescence test with standard methods for detection ofHelicobacter pylori, i.e. culture, the urease activity test and histological examination of tissue sections, using 158 biopsy specimens, 30 specimens were positive in all methods and 64 negative. In the remaining cases comparison was not possible because either immunofluorescence (29 specimens) or the standard methods (16 specimens) gave ambiguous results. The direct immunofluorescence test may have potential as an alternative to standard methods, but further testing in a defined patient population is necessary.     

Development of a rapid PCR method for identification ofHelicobacter pylori in dental plaque and gastric biopsy specimens

A rapid and simple polymerase chain reaction (PCR) method was developed to detectHelicobacter pylori in gastric biopsy specimens and dental plaque samples. The primers were targeted to the 16S rRNA sequence ofHelicobacter pylori strain ATCC 43504. The system was found to have a theoretical detection level of 0.5 to 5Helicobacter pylori cells in a 5 l sample of dental plaque. In the absence of plaque, the detection level was even better: theoretically, 0.05 to 0.5Helicobacter pylori cells were detected in water suspension. However, this appeared to be due to the presence of free bacterial DNA in the culture used for the sensitivity determination. Thus, the actual sensitivity of the system was found to be fewer than fiveHelicobacter pylori cells, irrespective of the type of sample used. The method was then used to analyse 29 dental plaque and gastric biopsy specimens collected from patients with a history of recurrent peptic ulcer disease. Fourteen stomach specimens were positive forHelicobacter pylori when tested with the PCR method, while the respective figures with culture, histological examination and the urease test were 11, 12 and 9. No positive dental plaque samples were observed.     

Antigenicity of fractions ofHelicobacter pylori prepared by fast protein liquid chromatography and urease captured by monoclonal antibodies

The antigenicity ofHelicobacter pylori protein fractions separated by fast protein liquid chromatography size exclusion was investigated by EIA with sera from patients of well definedHelicobacter pylori status. The antigenic material ofHelicobacter pylori was confined to fractions 8 and 14 to 21. Urease containing fractions (14/15) and flagella containing fractions (17/18) were identified. Fraction 8 non-specifically bound human immunoglobulin as demonstrated by the binding ofHelicobacter pylori negative sera. The remaining fractions 14 to 21 when used individually as EIA antigens were 91–100 % specific, however fractions 16 to 19 showed a reduced sensitivity (78 %) compared with the acid extract (95 %). The urease fractions were 91 % sensitive. Purified urease antigen captured by antiurease monoclonal antibodies was 83 % sensitive and 93.3 % specific.     

Antibacterial properties of lansoprazole alone and in combination with antimicrobial agents againstHelicobacter pylori

The activities of various types of antiulcer agents againstHelicobacter pylori strains were determined by an agar dilution method. Among the compounds tested, benzimidazole proton pump inhibitors were found to have significant activity against this organism. The activity of lansoprazole was fourfold more potent than that of omeprazole and bismuth subsalicylate, with MICs ranging from 1.56 to 25 µg/ml. Exposure ofHelicobacter pylori to lansoprazole led to an extensive loss of viability as well as suppression of virulence factors such as motility, adhesiveness to epithelial cells and urease activity. The combination of lansoprazole with antimicrobial agents such as penicillins, cephalosporins, macrolides, tetracyclines, aminoglycosides, quinolones, metronidazole and bismuth subsalicylate generally had an additive effect on inhibition ofHelicobacter pylori growth.     

Culture ofHelicobacter pylori from gastric biopsies transported in biopsy urease test tubes

Gastric biopsy specimens of 57 consecutively observed dyspeptic patients were studied for the presence ofHelicobacter pylori by histological examination, biopsy urease test (BUT) and culture. For culture, biopsy samples were transported in both Stuart media and BUT tubes. All 15 isolates could be cultured from both Stuart and BUT tubes. Thus, if the main reason for culture ofHelicobacter pylori is for antimicrobial susceptibility testing, only positive BUT tubes need to be submitted. This would reduce both the expense and the number of biopsies needed.     

Infection with Helicobacter pylori and Leukocyte Response in Patients with Myocardial Infarction

 To test whether Helicobacter pylori may contribute to the inflammatory response following myocardial infarction, the levels of IgG antibodies to Helicobacter pylori and some parameters of leukocyte activity were measured in 63 patients and 61 comparable controls. Helicobacter pylori-positive patients showed a significantly higher expression of the adhesion molecule LFA-1 on neutrophils than Helicobacter pylori-negative patients (433±29.0 vs. 398.8±38.9 mean fluorescence channels; P<0.0001), whereas no significant difference for any parameters tested was found in control subjects. These data suggest a role of Helicobacter pylori in inducing a leukocyte response following myocardial infarction.     

Serological Methods for Diagnosis of Helicobacter pylori Infection and Monitoring of Eradication Therapy

 Several methods can be used to diagnose Helicobacter pylori infection. Invasive methods include detection of the bacterium in gastric biopsy specimens by culture, immunohistochemistry, rapid urease tests, or the polymerase chain reaction. Noninvasive or less invasive detection methods include the urea breath test and serological methods. The urea breath test is based on the detection of ¹³CO₂ or ¹⁴CO₂ in breath, produced by bacterial urease in the stomach after labelled urea is swallowed. Serological methods are based on the detection of Helicobacter pylori-specific antibodies in serum, saliva, or urine. In this review, the performance and diagnostic value of several serological methods, such as enzyme immunoassay, rapid office-based assays, and Western blot, will be discussed in relation to biopsy-based methods and the urea breath test. In addition, the value of serological assays for monitoring eradication of Helicobacter pylori infection following treatment will be discussed. The diagnostic performance of properly evaluated serological assays is comparable to that of biopsy-based methods and the urea breath test. To monitor eradication of Helicobacter pylori infection following therapy, quantitative enzyme immunoassays can be used, especially in patients with high pretreatment antibody titres.     

Use of a receiver operating characteristic in the evaluation of two commercial enzyme immunoassays for detection ofHelicobacter pylori infection

Two novel commercial IgG enzyme immunoassay (EIA) systems based on acid-glycine-extracted (Pyloriset IgG EIA, Orion Diagnostica) or fast protein liquid chromatography-purified (Cobas Core Anti-H. pylori EIA, Roche Diagnostic Systems)Helicobacter antigens were evaluated in a prospective study involving 127 patients. All patients underwent upper endoscopy with biopsy, and biopsies were examined for the presence ofHelicobacter pylori by a rapid urease test, microscopy and culture. Of the 71 patients found to be infected withHelicobacter pylori, 69 (97.2 %) and 65 (91.5 %) tested positive with the Cobas Core and Pyloriset test, respectively. A detailed receiver operating characteristic analysis of the two tests showed that the Cobas Core assay was more sensitive and specific at every possible cut-off level; gave a better resolution of individual results, indicating a greater fine-sensitivity; and had no grey zone compared to a large grey zone encompassing 13.4 % of the serum samples tested with the Pyloriset EIA. The Cobas Core assay appears to be a valuable tool for epidemiological purposes as well as for pre-endoscopic screening of dyspeptic patients.     

Serodiagnosis ofHelicobacter pylori infections with an enzyme immunoassay using the chromatographically purified 120 kilodalton protein

A membrane-associated 120 kDa protein ofHelicobacter pylori with known species-specificity was isolated and used in an enzyme immunoassay (EIA) for the detection ofHelicobacter pylori-specific IgG antibodies in patient sera. The EIA was compared with two other methods used for serodiagnosis ofHelicobacter pylori infections: an EIA using sonicated wholeHelicobacter pylori cell antigen and Western immunoblot. In a prospective study 127 unselected patients (76 patients with antrum gastritis, 51 patients without gastritis) who underwent gastroscopy were studied histologically and serologically. The EIA using the purified 120 kDa protein had the highest specificity (92 %) compared with the EIA using a whole cell sonicate of a singleHelicobacter pylori strain as antigen (60.7 %) and the immunoblot (90.2 %). The sensitivity was 96 %, 100 % and 92 %, respectively. Sera of three control patients reacted strongly in all three methods, indicating possibleHelicobacter pylori infection with negative histological findings. The EIA using the 120 kDa protein as antigen was shown to be a specific and sensitive technique for the serodiagnosis ofHelicobacter pylori infections.     

Effect of specimen collection techniques,transport media,and incubation of cultures on the detection rate ofHelicobacter pylori

Culture and histologic examination are considered gold standard methods for the detection ofHelicobacter pylori, but discrepancies may occur with either method. Failure to detectHelicobacter pylori may be due to sampling error, inappropriate transport or culture media, or insufficient duration of the incubation period. Rates of detection ofHelicobacter pylori by culture and histopathologic examination of gastric mucosal biopsy specimens were determined in 102 consecutive dyspeptic patients. In a separate group of 60 patients, rates of detection ofHelicobacter pylori by culture of antral brushings and the length of incubation required in selective and nonselective culture media were studied. In the first group of 102 patients, the combination of culture and histologic examination detected 54Helicobacter pylori-positive patients, whereas the separate techniques each detected 51Helicobacter pylori-positive patients. In the second group of 60 patients evaluated by culture of antral brushings, the rate of detection ofHelicobacter pylori was 25 of 60 and was similar for culture (25/60) and histologic examination (25/60). In the second group the length of incubation required to detectHelicobacter pylori was different for selective and nonselective media. In nonselective media, incubation of up to ten days was required to detect allHelicobacter pylori infections, whereas in selective media seven days was sufficient. Rates of detection ofHelicobacter pylori by culture, histopathologic examination and culture from brushings were similar, whereas the combination of culture and histopathologic examination achieved a superior rate of detection. The incubation period required for the detection ofHelicobacter pylori by culture was a minimum of seven days and was dependent on the culture medium used.     

Evaluation of a serological test for diagnosis ofHelicobacter pylori infection in children

A serological test for the diagnosis ofHelicobacter pylori infection (Cobas Core Roche, IgG, 2nd Generation; Roche, France) was compared with the examination of biopsy samples (culture and histology) obtained after endoscopy in 115 children to assess its value. In 94 children (42 positive and 52 negative), results were concordant. In 10 children a positive serological test was associated with an absence ofHelicobacter pylori, while in 11 others a negative serological test was associated with a positive culture. Sensitivity of the test was 79.2% and specificity 83.9%. A relationship between IgG titers and age (r=0.31, p<0.05) was found. Serological tests could be useful for the diagnosis ofHelicobacter pylori infection, but a negative test result does not rule out infection, particularly in children under 10 years of age.     

Evaluation of a commercial latex test for serological diagnosis ofHelicobacter pylori infection in treated and untreated patients

The value of a commercially available latex test (Pyloriset) for the diagnosis ofHelicobacter pylori infection by demonstration of specific antibodies was compared with that of direct diagnostic methods such as culture, biopsy-urease test and microscopy of fuchsin-stained smears. The sera were from 136 patients who prior to this study either had or had not been treated forHelicobacter pylori-infection simultaneously with amoxicillin (3 × 750 mg/day) and metronidazole (3 × 500 mg/day) for 12 days. On average, the sensitivity of the test was 90 %. The specificity with sera from untreated patients was 75.9 %; with sera from treated patients specificity was 22.2 %, 28 % and 20 % 1, 3 and 6 months respectively after start of treatment. Only as late as one year after the onset of chemotherapy did the specificity return to 67 %. Because of its low specificity this test does not offer any advantage over other tests in the detection ofHelicobacter pylori-infection or in monitoring the chemotherapeutic success.     

Use of a urea breath test versus invasive methods to determine the prevalence ofHelicobacter pylori in zaire

The prevalence ofHelicobacter pylori infection in Zaire was determined by means of a [¹⁴C] urea breath test in 133 asymptomatic subjects, by culture and histological examination of biopsies in 324 consecutive endoscopy patients with chronic epigastric complaints, and by both the breath test and culture/histology in a subset of 92 patients. Sixty healthy Belgian students or hospital laboratory workers were also included for comparison. The prevalence ofHelicobacter pylori was significantly higher in asymptomatic Zairian subjects (77.4 %) than in the Belgians (30 %; p<10^–6). Infection was also acquired much earlier in life in Africans, 66% of the children aged 5 to 9 years already being infected versus none of the Belgian subjects below the age of 20 years. In Zaire, however, the prevalence ofHelicobacter pylori in patients with gastroduodenal disorders (87.5 %) was similar to that in the group of asymptomatic subjects (77.5 %) after adjustment for age and other epidemiological parameters (gender, place of residency, education level, smoking and drinking habits) in a multivariate analysis. The high rate of acquisition ofHelicobacter pylori infection in Zaire emphasizes the need to consider the baseline prevalence ofHelicobacter pylori in a defined population when studying its association with various diseases.     

Detection ofHelicobacter pylori in gastric biopsy tissue by polymerase chain reaction

To evaluate the sensitivity of a polymerase chain reaction (PCR) assay using nested primers in detectingHelicobacter pylori, gastric tissue biopsy specimens were collected on endoscopy from 17 patients with a duodenal ulcer. DNA was extracted by phenol/chloroform treatment or boiling in water, and then subjected to a nested PCR using two primer pairs from the urease gene ofHelicobacter pylori. Fourteen of the 17 patients were positive forHelicobacter pylori using DNA samples extracted by either method. The PCR results correlated well with the results of an enzyme immunoassay to detect IgG antibody. However, there were two culture negative patients. The three PCR negative patients were both culture negative and serologically negative. DNA from 9 of the 14 patients was randomly selected and subjected to semiquantification by serial dilutions, and then PCR. The results showed that phenol/chloroform extraction yielded 10–1000 times more DNA than the boiling method. It is concluded that the PCR assay is a rapid and sensitive method for detectingHelicobacter pylori, and that phenol/chloroform extraction is superior to simple boiling in obtaining DNA samples for PCR.     

Transport conditions and number of biopsies necessary for culture ofHelicobacter pylori

Stuart's transport medium with a charcoal impregnated swab was tested for transport of biopsies from the gastric antrum for culture ofHelicobacter pylori. Biopsies were cultured under microaerophilic conditions either within 2 h or after a delay of 24 h at 4 °C. In 65 patients referred for gastroscopy two biopsies were taken.Helicobacter pylori was found in 39 patients. The rate of survival ofHelicobacter pylori was found to be as high in Stuart's transport medium after 24 h at 4 °C as in the paired biopsy which was cultured immediately after transportation in normal saline. In five (13 %) of 39 patients positive forHelicobacter pylori, the organism was cultured from only one of the biopsies. It is therefore recommended that two biopsies be taken for culture.     

Evaluation of a Single-Step Serological Assay for Laboratory Diagnosis of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Infection

A rapid, single-step, in-laboratory qualitative test for the detection of IgG antibodies to Helicobacter pylori in serum (TestPack Plus; Abbott Laboratories, Germany) was evaluated. This test may be used as an alternative to enzyme immunoassays (EIAs). Of 153 adult patients, 110 were defined as Helicobacter pylori positive and 43 as Helicobacter pylori negative by the gold standard, a combination of three tests. The performance characteristics of the TestPack Plus, i.e. sensitivity, specificity, and positive and negative predictive values, were not significantly different from the corresponding values obtained with an EIA used for comparative purposes, the Pyloriset EIA-G test (Orion Diagnostica, Finland). The high positive predictive value (93%) of the TestPack Plus single-step serological test makes it a valuable tool for rapid in-laboratory screening purposes, especially in countries with a high prevalence of Helicobacter pylori infection. Electronic Publication