Detection of Helicobacter pylori antigen in stool by enzyme immunoassay

Invasive techniques for diagnosis of Helicobacter pylori (H. pylori) infection require an endoscopic examination which is expensive and inconvenient and may cause complications. Stool cultures for H. pylori or a direct detection of H. pylori antigen in stools by PCR are expensive, tedious, and have a low sensitivity. We recently used an enzyme immunoassay (EIA) to detect H. pylori antigen in stool specimens. A total of 41 patients were seen at Inha University Hospital, Inchon, Korea between September and October 1998. There were 26 men and 15 women who had an average age of 37.6 years which ranged from 5 to 71 years in the present study. All of these patients came to the hospital complaining of an upper abdominal discomfort and were subjected to endoscopy and biopsies. Fifteen had a gastric ulcer, 13 had a duodenal ulcer, 1 had an early gastric cancer, and there were 12 chronic gastritis patients as shown by endoscopy. The biopsy specimens were examined by histology, CLO test, and cultures and these results were used as gold standards. Stool specimens were tested for the H. pylori antigen by EIA. A dual wavelength cut-off of 0.100 that was recommended by the manufacturer gave a good performance (87.1% sensitivity, 100% specificity, 100% positive predictive value, 71.4% negative predictive value, and a 90.2% efficiency). But the adjusted cut-off value using the receiver operating characteristic curve improved the performance of the test (using the cut-off value of 0.024, the sensitivity, specificity, PPV, NPV, and efficiency were 100%, 90.0%, 96.9%, 100%, and 97.6% respectively). Re-evaluation of the cut-off value may be needed for Korean patients. This technique is non-invasive, rapid, easy-to-use, and shows good performance characteristics for diagnosis of H. pylori infections. Therefore, this technique may be a substitute for gastric endoscopy especially in children and some patients who are unable to tolerate an endoscopic examination and it may be substituted for a serologic test in epidemiological research.     

幽门螺杆菌粪便抗原检测方法学评价

目的 评价幽门螺杆菌粪便抗原（ HpSA）检测在诊断患者幽门螺杆菌（Hp）感染中的价值.方法 以胃镜病理学诊断结果为金标准,对328例有消化道症状患者粪便,同时应用酶联免疫吸附实验检测幽门螺杆菌粪便抗原,计算HpSA检测诊断Hp感染的特异性、灵敏度和准确性等性能指标.结果 幽门杆菌粪便抗原酶免疫检测法对Hp感染诊断与金标准相比无统计学差异（P＞0.05）,其敏感性为94.6％、特异性为96.9％、准确性为96.3％、阳性预期值为89.7％、阴性预期值为98.4％.结论 幽门螺杆菌粪便抗原酶免疫检测是一种简便、易行、准确性高、便宜、易重复的非侵入性检测方法.     

Significance of transiently positive enzyme-linked immunosorbent assay results in detection of Helicobacter pylori in stool samples from children

In young children, the significance of stool samples transiently positive for Helicobacter pylori antigen is unknown. As part of a larger prospective study on enteric infections, stool samples were obtained from 323 children at two time points 3 months apart and tested for H. pylori antigen using a commercially available enzyme-linked immunosorbent assay (ELISA) test. Seminested PCR for a Helicobacter-specific 16S rRNA gene was performed on all 26 pairs reverting from positive to negative (transient positives), all 4 persistent antigen-positive pairs, and 10 randomly selected persistent antigen-negative pairs. Helicobacter species were amplified from the first stool samples of 15/26 (58%) of the transient positives and 1 (25%) of 4 persistent positives. No Helicobacter species were amplified from the 10 persistent negatives. Among the 15 amplicons from transient-positive stool, H. pylori was sequenced and identified from 12 (80%; 95% confidence interval, 52% to 96%) and other Helicobacter spp. were identified from three (Helicobacter canis, Helicobacter winghamensis, and MIT 99-5504). Four of the 15 remained positive by PCR for the second (antigen-negative) stool sample, including all 3 initially identified as non-H. pylori. Helicobacter bilis was amplified from the second sample of a persistent positive. Two of eight transient positives from whom serum was available had accompanying transient elevations in anti-H. pylori antibodies. Transiently positive stool ELISAs for H. pylori are common and represent H. pylori in the majority of cases where sequences can be obtained. A not-insignificant percentage of antigen-positive stools, however, may represent other Helicobacter species.     

Characteristics and trends of clarithromycin-resistant Helicobacter pylori isolates in Japan over a decade.

Clarithromycin has been administered to patients in Japan since 1991. Clarithromycin-resistant Helicobacter pylori strains have been on the rise in Japan. We obtained H. pylori isolates between 1989 and 2000 and examined mutations of the 23S rRNA gene, which are closely associated with clarithromycin resistance. Isolates were obtained from 356 patients with H. pylori infection treated at the Hiroshima University. Sixty-one of the patients received clarithromycin-based H. pylori eradication therapy. Mutations of the 23S rRNA gene were examined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by sequencing analysis. Mutant strains were found in 42 of the 356 patients (11.8%). The prevalence of mutant strains increased from 0 to 20.4% during the 12-year study period. The prevalence increased to more than 10% by 1995 and then to more 20% after 1999. The H. pylori eradication rate was significantly higher in patients with wild-type strains than in patients with mutant strains (72.0 vs. 36.4%, p = 0.024). Our data indicate that clarithromycin-resistant H. pylori strains have increased rapidly since 1995 and that the effectiveness of clarithromycin-based H. pylori eradication therapies may soon be compromised. Other new therapies may be necessary as first-line treatments in Japan.     

Intrafamilial spread of the same clarithromycin-resistant Helicobacter pylori infection confirmed by molecular analysis

Clarithromycin-resistant Helicobacter pylori (CRHP) is increasing worldwide, especially in children. We report a family case in which both the mother and child were infected with CRHP. DNA analysis revealed that all of the mother's and daughter's isolates were indistinguishable, suggesting that the same CRHP strain spread between the family members. The spread of CRHP within families may be increasing.     

Comparison of three stool antigen tests for Helicobacter pylori detection

BACKGROUND: Active Helicobacter pylori infection can be diagnosed by invasive (biopsy based) or non-invasive methods, such as stool antigen testing. AIMS: To compare three stool antigen enzyme immunoassay kits--Premier Platinum Hp SA, FemtoLab Cnx, and Hp Ag--with biopsy based methods for the detection of H pylori in previously undiagnosed patients. METHODS: One hundred and eleven adults with dyspepsia referred for endoscopy provided a stool sample for testing and had biopsies taken. Patients were considered H pylori positive if two out of three invasive tests were positive or if culture alone was positive. RESULTS: The sensitivities and specificities of the Premier Platinum Hp SA, FemtoLab Cnx, and Hp Ag stool antigen kits when compared with biopsy based diagnosis were, 63.6%, 88.0%, and 56.0% and 92.6%, 97.6%, and 97.6%, respectively. CONCLUSIONS: FemtoLab Cnx may be considered as an alternative to urea breath testing in the initial diagnosis of patients with dyspepsia who do not require immediate endoscopy. Stool testing has the potential advantages of being simple to perform, relatively cheap, and samples can be submitted directly from primary care.     

Detection of clarithromycin-resistant helicobacter pylori strains by a preferential homoduplex formation assay

It has been shown that resistance to clarithromycin, a major cause of failure in Helicobacter pylori eradication therapy, is associated with point mutations in the 23S rRNA gene. We sought to apply the preferential homoduplex formation assay (PHFA), a novel technique for the efficient detection of point mutations, to detection of the mutations. PHFA was performed on streptavidin-coated microtiter plates with biotin- and dinitrophenyl-labeled amplicons to detect the wild-type gene or each mutant gene. DNA samples were extracted from gastric juice specimens of 412 patients with H. pylori infection and were applied to the assay. The detection threshold of PHFA was as few as 10 gene copies. The sensitivity of PHFA for the detection of H. pylori infection was higher than those of culture and the rapid urease test. A total of 337 (81.8%) samples had the wild-type gene, 38 (9.2%) had the A2144G mutation, and 37 (9.0%) contained both the wild type and a mutation (A2144G in 30 samples, A2143G in 5 samples, and A2143G plus A2144G in 2 samples). About half the strains isolated from patients with mixed infection were susceptible by the agar dilution method (MIC, <0.1 mg/liter). Therefore, PHFA can detect clarithromycin-resistant H. pylori strains, even in patients with mixed infections with the wild type, that are not detectable by the agar dilution method.     

Evaluation of a novel rapid one-step immunochromatographic assay for detection of monoclonal Helicobacter pylori antigen in stool samples from children

A new rapid one-step immunochromatographic test using monoclonal antibodies for detection of Helicobacter pylori antigen in stool in children was evaluated on coded stool samples from 159 children (mean age, 9.7 +/- 5.0 years; 118 from Munich, 41 from Vienna): 86 children were H. pylori infected defined by positive culture and/or > or =2 other positive tests ([13C]urea breath test, histology, rapid urease test), and 73 children showed concordant negative results. Seventy-nine patients (12.1 +/- 3.8 years; 42 from Munich; 37 from Vienna) were tested 6 to 8 weeks after anti-Helicobacter pylori therapy with urea breath test and stool test. In Munich, all 160 tests (118 pre- and 42 posttreatment) were independently read by two observers. Equivocal results were excluded for calculation of sensitivity and specificity but were considered as false to assess accuracy. The two observers in Munich agreed in 63 out of 65 positive and 89 out of 95 negative results, while eight times (5.0%) they judged the test as equivocal. Pretreatment and posttreatment results for sensitivity were 88.1% (79.2 to 94.1) and 88.9% (51.8 to 99.7), specificity 88.1% (77.8 to 94.1) and 93.9% (85.2 to 98.3), and accuracy 83.5% and 81.5%, respectively. We conclude that the new monoclonal immunochromatographic quick test shows a good interobserver agreement, but equivocal results occur in 5%. Performance is comparable before and after therapy. The test may become a good alternative in children in settings where a [13C]urea breath test or a reliable enzyme immunoassay stool test are not available.     

Detection of Blastocystis from stool samples using real-time PCR

We developed a real-time LC PCR assay to detect a 152 bp sequence in an uncharacterized region of the Blastocystis genome. The described assay detected 11 of 11 ATCC strains of Blastocystis from subtypes 1, 3, and 4. Three of three stool samples from Oregon and California military personnel that were negative for Blastocystis by an ova and parasite test as well as a conventional PCR assay were positive for Blastocystis using our real-time LC PCR assay. Diagnosis of Blastocystis infections using this sensitive method, including DNA extraction and real-time PCR, only requires 3 h. The lower limit of detection for Blastocystis in stool using the real-time LC PCR assay was calculated to be 760 cells of Blastocystis per 100 mg of stool, an estimated 760 parasites per reaction. The assay did not cross-react with Ruminococcus hansenii, Anarococcus hydrogenalis, Bifidobacterium adolescentis, Fusobacterium prausnitzii, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, or Lactobacillus acidophilus. Because of the ease of use, sensitivity, specificity, and increase in Blastocystis infections in the USA we believe this assay has the potential to be useful as a clinical diagnosis tool of Blastocystis infection.     

Posttreatment follow-up of Helicobacter pylori infection using a stool antigen immunoassay.

The Helicobacter pylori stool antigen enzyme immunoassay (HpSA) was evaluated during posttreatment follow-up of patients in a country with a very high prevalence of H. pylori infection. From among 273 dyspeptic individuals (18 to 55 years) initially recruited from a shantytown in Lima, Peru, 238 participants who met the inclusion criteria and were suspected to be H. pylori positive based on (14)C urea breath test (UBT) results underwent endoscopy. Participants with endoscopy-proven infections received standard eradication therapy and were monitored by UBT and HpSA at 1 month following treatment and at 3-month intervals for 9 months posttreatment. A second endoscopy was performed if UBT results showed evidence of treatment failure or H. pylori recurrence. Biopsy results were considered the "gold standard" in all analyses. Among patients who underwent endoscopy, HpSA had a pretreatment sensitivity of 93%. Two-hundred thirty patients completed the treatment regimen, of whom 201 (93%) were considered to have had successful treatment outcomes based on a negative follow-up UBT. Thirty-two patients with UBT-defined treatment failures or H. pylori recurrences at any point during the 9-month follow-up underwent a second endoscopy. In the posttreatment setting, HpSA had an overall sensitivity of 73% and a specificity of 67%. Agreement between UBT and HpSA diminished throughout the follow-up. Among 14 participants in whom HpSA remained positive at 1 month following treatment despite UBT evidence of treatment success, 12 (86%) became HpSA negative within 3 months posttreatment. Although this study confirmed the validity of the HpSA in the initial assessment of dyspeptic patients, the test demonstrated a reduced overall accuracy in the detection of treatment failures and H. pylori recurrences during 9 months of posttreatment follow-up. Furthermore, in some patients it may take up to 3 months after successful eradication for antigen shedding to diminish to levels within the negative HpSA range.     

Detection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluorescent in situ hybridization

We report on the successful application of fluorescent in situ hybridization for detection of Helicobacter pylori and determination of its clarithromycin susceptibility in formalin-fixed and paraffin-embedded gastric biopsy specimens that had been prepared for pathological examination. This method is useful when results from conventional culturing with antibiotic susceptibility testing are not available.     

在兰州地区儿童粪便中检测Aichi virus

目的 从兰州腹泻和正常儿童粪便标本中检测 Aichi virus,同时探讨 Aichi virus 与婴幼儿腹泻之间的联系.方法 根据文献发表资料,采用RT-PCR方法扩增 Aichi virus 3CD 片段,阳性产物经测序确定,并与已发表的该病毒序列进行比对分析.结果 在46份腹泻住院患儿粪便标本和299份腹泻门诊就诊患儿标本中各检出1例 Aichi virus,总检出率为0.06%,正常对照儿童中未检测到 Aichi virus.2株病毒3CD区基因与已知参考株核苷酸序列同源性均为97%,系统进化分析显示这2株病毒属于B基因型.结论 我国存在B基因型的 Aichi virus,但要明确我国 Aichi virus 的病原学及流行病学特点需要更多研究.     

Detection of Helicobacter pylori infection in symptomatic Bulgarian adults

This study assessed the prevalence of Helicobacter pylori in symptomatic Bulgarian adults by means of culture, Gram's stain and an in-house rapid urease test (RUT), and also assessed the H. pylori density by culture. In total, 1441 non-treated and 270 treated patients were evaluated. Most non-treated patients with ulcers (87.7%), gastric malignancy (79.2%) and other gastroduodenal diseases (73.4%) were H. pylori-positive. Among non-treated and treated patients, 75.3% and 54.8%, respectively, of elderly patients, and 78.3% and 56.1%, respectively, of other adults were H. pylori-positive. Two (0.1%) non-treated adults were Helicobacter heilmannii-positive. The accuracy of direct Gram's stain and the in-house RUT were 74.8% and 64.2% in non-treated patients, and 73.7% and 63.0% in treated patients, respectively. Culture was highly accurate (>95%) in both groups. Older age decreased the sensitivity of the RUT in non-treated patients by 10.7% and that of all tests in treated patients by 6.9-8.1%. Incubation for 11 days was required for the growth of 2% and 4% of the strains from treated patients on selective and non-selective medium, respectively. There were no differences in isolation rates between positive fresh (74.2%) and frozen (75.2%) specimens. In non-treated adults, a high H. pylori density (growth in all quadrants of the plates) was more common (43.1%) in ulcer patients than in other patients (25.4%). In conclusion, H. pylori infection was common in Bulgarian patients, and at a high density in >40% of ulcer patients, while H. heilmannii infection was uncommon. Culture provided a highly accurate diagnostic approach. Stomach biopsies from non-treated patients can be frozen for several days. The benefit of reporting H. pylori density, as determined by culture, requires further evaluation.     

Immunomagnetic separation and PCR for detection of Helicobacter pylori in water and stool specimens.

The detection of Helicobacter pylori in clinical and environmental samples by PCR sometimes requires removal of polymerase inhibitors. We have used a magnetic immunoseparation technique as pre-PCR treatment to facilitate direct detection of H. pylori in stool and water specimens. Rabbit hyperimmune antiserum was produced and magnetic beads were coated with purified immunoglobulin G, which reacted with and bound to both coccoid and rod-shaped forms of H. pylori. When PCR was applied for the detection of H. pylori from cultured samples, the number of organisms that was required for positive scores varied significantly. For a 3-day culture of H. pylori, samples containing 10(2) bacteria per ml are needed for a positive score; for a 6-day culture, samples containing 10(4) bacteria per ml are needed; and for a 10-day culture, samples containing 10(6) bacteria per ml are needed. These results indicate that the coccoid forms of H. pylori may have a different antigenicity and DNA content and are therefore more difficult to detect by immunomagnetic separation and PCR than the rod-shaped forms. Spiked samples with the addition of feces, spiked water samples, and a patient stool specimen were all scored positive with this technique.     

Detection ofGiardia lamblia cysts in stool samples by immunofluorescence using monoclonal antibody

The diagnostic potential of indirect immunofluorescence to detectGiardia cysts in stool samples using a cyst-specific anti-Giardia lamblia monoclonal antibody was evaluated in comparison to conventional light microscopy. One hundred fifty specimens from clinically suspectedGiardia infections and 50 control samples from microscopically provedGiardia infections were tested.Giardia cysts were found in 15 of 150 (10 %) samples tested by light microscopy, whereas immunofluorescence microscopy detected 35 of 150 (23 %) positive samples. Forty-six of the 50 reference samples previously shown to containGiardia cysts were positive. Apparently, the four discrepant samples contained very low numbers of parasites, as none could be detected by conventional microscopy. The results show thatGiardia lamblia cysts are detected significantly more frequently using the antibody marker. The doubled number of positive stool specimens and detection of as little as four cysts per sample suggest that microscopical examination of samples can be improved by immunofluorescent staining ofGiardia lamblia cysts.