Transport and Storage of Fresh and Frozen Gastric Biopsy Specimens for Optimal Recovery of Helicobacter pylori

The viability of Helicobacter pylori in vitro and in gastric biopsy specimens was determined. Recovery rates were 94, 87, and 77% from biopsy specimens in Portagerm pylori in cooled containers after 1, 2, and 3 days of transport, respectively (n = 307), and 97% if stored and shipped in glycerol broth at -70 degrees C (n = 232).     

A Novel Method for Genotyping the Helicobacter pylori vacA Intermediate Region Directly in Gastric Biopsy Specimens

The present report describes a novel method for genotyping the virulence-associated vacA intermediate (i) region of Helicobacter pylori in archive material. vacA i-region genotypes as determined by the novel method were completely concordant with those of sequence analysis and with those of functional vacuolation activity. The method was further validated directly in gastric biopsy specimens of 386 H. pylori-positive cases, and effective characterization of the vacA i region was obtained in 191 of 192 (99.5%) frozen and in 186 of 194 (95.9%) formalin-fixed paraffin-embedded gastric biopsy specimens, respectively. The genotyping method was next used to address the relationship between the vacA genotypes and the cagA status. The vacA i1 genotype was associated with vacA s1 (where s indicates signal region), vacA m1 (where m indicates middle region), and cagA-positive genotypes (P < 0.0001), while the vacA i2 genotype was closely related with vacA s2, vacA m2, and cagA-negative genotypes (P < 0.0001). The relationship between H. pylori vacA i-region genotypes and gastric disease development was subsequently evaluated in the Portuguese population. Patients infected with vacA i1 strains showed an increased risk for gastric atrophy and for gastric carcinoma, with odds ratios of 8.0 (95% confidence interval [CI], 2.3 to 27) and of 22 (95% CI, 7.9 to 63), respectively. Taken together, the results show that this novel H. pylori vacA i-region genotyping method can be applied directly to archive material, providing a fast evaluation of strain virulence determinants without the need of culture. The results further emphasize that the characterization of the vacA i region may be useful to identify patients at higher risk of gastric carcinoma development.     

Clarithromycin-Susceptible and -Resistant Helicobacter pylori Isolates with Identical Randomly Amplified Polymorphic DNA-PCR Genotypes Cultured from Single Gastric Biopsy Specimens Prior to Antibiotic Therapy

Of the Helicobacter pylori populations from 976 patients, six contained clarithromycin-resistant as well as -susceptible colonies. In each heterogeneous H. pylori population, resistant H. pylori colonies harbored identical 23S ribosomal DNA (rDNA) mutations associated with clarithromycin resistance, while the susceptible H. pylori colonies all had wild-type 23S rDNA. The resistant and susceptible colonies of each of the heterogeneous H. pylori populations had identical randomly amplified polymorphic DNA-PCR genotypes. In conclusion, evaluation of antimicrobial susceptibility can be misinterpreted if only a single colony from the primary H. pylori population is used to test for clarithromycin susceptibility.     

New Transport Medium for Cultural Recovery of Helicobacter pylori

We developed a new transport medium (GESA—Helicobacter pylori transport medium [publication no. WO/2014/019696, patent pending no. PCT/EP2013/002292; Liofilchem s.r.l., Roseto degli Abruzzi, Teramo, Italy]) for recovery of Helicobacter pylori from gastric biopsy samples. GESA transport medium, in a semisolid state, provides the optimal conditions for maintaining the viability of the microorganism over time. The efficacy of the transport medium was assessed through in vitro and ex vivo experiments. We were able to recover different suspensions of H. pylori ATCC 43629 and H. pylori 13 A in GESA transport medium stored at 4°C for up to 10 days. In particular, with a starting inoculum of ∼10⁵ CFU, after 7 days of storage, 150 ± 25 CFU and 40 ± 7 CFU of the reference and clinical strains were detected, respectively. H. pylori colonies were isolated from gastric specimens taken from both the antrum and the fundus in 68 (90.66%) of 75 urea breath test (UBT)-positive patients. Moreover, GESA transport medium allowed the recovery and isolation of H. pylori colonies from additional biopsy samples from 13 of the 75 detected subjects at up to 10 days of biopsy sample storage at 4°C. Finally, GESA transport medium preserved its characteristics when stored at 4°C for 1 year from its preparation, thus allowing good recovery of H. pylori. GESA transport medium can be considered a standardized transport medium with high performance that optimizes the recovery rate of H. pylori grown by culture.     

Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n = 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n = 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin-resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that cells of different H. pylori strains can subsist in very close proximity in the stomach.     

Prevalence and Genotypes of Helicobacter pylori in Gastric Biopsy Specimens from Patients with Gastroduodenal Pathologies in the Cukurova Region of Turkey

The effects of Helicobacter pylori genotypes on clinical prognosis in the Cukurova region of Turkey were investigated by PCR. The prevalence of type I strains carrying the s1c allele, unlike in neighboring regions and countries, was found to be significantly higher in patients with gastritis and/or gastric ulcers (P = 0.001), and that of type I strains carrying the s1a allele was found to be significantly higher in patients with duodenal ulcers (P < 0.001). The cagA gene was strongly associated with the more virulent vacA genotypes (P < 0.001).The effects of vacA allelic combinations of Helicobacter pylori on clinical prognosis show geographic differences in Western and Eastern populations. Although all strains of H. pylori contain the vacA gene, they vary in the ability to produce cytotoxin. Type m1 strains demonstrate more toxin activity than m2 strains, type s1a is more active than s1b, and type s2 produces no detectable activity (2, 3). The s1a alleles are frequently observed in strains from northern and eastern Europe, while the s1b allelic types are frequent in such regions as Central and South America, Spain, Portugal, and South Africa. The s1b types are rare in other regions and less virulent than other s1 subtypes (2, 3, 10, 19, 20, 22, 23). Furthermore, although the s1c allele has been stated to be more common in Asian strains and it may be associated with gastric cancers that are prevalent in that region, other studies have, in contrast, indicated that cancer development cannot be explained by s1c alleles alone (4, 12, 18-20, 23). Thus, the relationship between the genotypes of causative strains and clinical outcome should be considered in different geographic regions in order to make a true estimation of prognosis. There are very few studies on the relationship of vacA alleles with clinical outcomes in Turkey, and no such study has been carried out in our region (5, 6, 15). Therefore, we aimed to determine the prevalence of H. pylori, the effects of the cagA and vacA genes and vacA allelic types of the colonizing strains on clinical prognosis, and the relationship between the cagA and vacA genotypes in patients with gastritis and/or gastric ulcers (G/GU) and those with duodenal ulcers (DU) in the Cukurova region of Turkey.(Part of this study was presented as a poster [Determination of the Prevalence and Genotypes of H. pylori in Gastric Biopsy Specimens from Patients with Gastroduodenal Pathologies] at the XXI International Workshop on Helicobacter and Related Bacteria in Chronic Digestive Inflammation and Gastric Cancer in Riga, Latvia, 18 to 20 September 2008, and an abstract was published [Helicobacter 13:420, 2008].)The subjects of this study were 231 previously untreated patients seen at outpatient clinics of the gastroenterology departments of two regional hospitals between January 2005 and January 2006, 158 of whom had endoscopically diagnosed G/GU and 73 of whom had DU. Antral gastric biopsy specimens were transported to the laboratory in brain heart infusion broth (Oxoid, Basingstoke, Hampshire, England) within 2 h and stored at −20°C.Extraction of DNA was performed with a QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer''s instructions. The extracted DNA was measured in an Optimum-One UV-VIS spectrophotometer (Chebios, Rome, Italy) at a 260-nm wavelength for determination of DNA concentrations, which were confirmed to be sufficient for amplification (>3 μg/ml). Then, the DNA samples were stored at −20°C until the amplification of all of the gene regions to be investigated was completed.These samples were first examined by glmM (formerly ureC) gene PCR, the most sensitive, specific, and reliable method for determination of the prevalence of H. pylori (11, 13). H. pylori-positive DNA samples were analyzed for the cagA and vacA genes, and vacA-positive DNA samples were further investigated for the detection of vacA alleles (s1a, s1b, s1c, and s2 in the signal region and m1a, m1b, and m2 in the cytotoxin-encoding middle region) by the PCR method. The primers used in this study are shown in Table ​Table1.1. All amplification procedures were performed with a Thermal Cycler 2720 (Applied Biosystems, Foster City, CA) with a total volume of 50 μl of a PCR mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM each deoxynucleoside triphosphate, 25 pmol of each primer, 2.5 U of Taq polymerase, and 5 μl of each DNA sample. The PCR cycling conditions were as described previously (2, 13, 22, 23). Amplified PCR products were examined by electrophoresis on 2% agarose gels with ethidium bromide, and bands were analyzed under UV illumination.

TABLE 1.

Oligonucleotide primers used in this study

胃镜活检标本诊断胃癌的临床病理分析

目的探讨胃镜活检标本诊断胃癌的临床病理特征。方法收集123例胃镜活检并经手术切除标本证实为胃癌的病例,观察分析组织形态学结构。结果胃恶性肿瘤以腺癌为主,且癌性腺体浸润至粘膜下层52例,癌性腺体在粘膜固有层内浸润32例,印戒细胞癌28例,淋巴瘤5例,间质瘤6例。结论胃镜活检标本诊断胃癌在腺癌中要紧密结合癌细胞形态特征及癌性腺体的形态特征,浸润性生长方式和浸润深度,纤维结缔组织间质反应,肿瘤性坏死及胃镜所见,其它组织学类型恶性肿瘤需结合镜下结构和临床病例资料。     

Use of a Gastric Juice-Based PCR Assay To Detect Helicobacter pylori Infection in Culture-Negative Patients

A gastric juice-based PCR assay was compared with culture, microscopy, and a rapid urease test with specimens from 114 subjects. The PCR and conventional tests were positive for 76 and 62% of the subjects, respectively. The prevalence of gastroduodenal disease and seropositivity for anti-Helicobacter pylori immunoglobulin G were similarly high among conventional-test-positive and PCR-only-positive subjects compared to all-negative ones. The PCR assay is recommended to confirm the H. pylori status of culture-negative peptic-ulcer patients.     

homB Status of Helicobacter pylori as a Novel Marker To Distinguish Gastric Cancer from Duodenal Ulcer

The hom family of Helicobacter pylori outer-membrane proteins, especially the homB gene, has been suggested as a novel virulence factor; however, the clinical association and function of this gene are still unclear. We evaluated the presence of the homA, homB, and cagA genes in 286 strains isolated from patients in the U.S. and Colombian populations (126 with gastritis, 96 with duodenal ulcer, and 64 with gastric cancer) by PCR. The results were compared with the clinical presentation and gastric injury. The prevalence of the homB gene was significantly higher in strains isolated from gastric-cancer patients (71.9%) than in those from duodenal ulcer patients (52.1%) (P = 0.012). In a multivariate analysis, the presence of the cagA gene significantly increased the risk for developing gastric cancer and duodenal ulcer, with the presence of the homB gene acting as a factor that could distinguish gastric cancer from duodenal ulcer (adjusted odds ratio, 3.033; 95% confidence interval, ∼1.37 to ∼6.73). cagA status was correlated with homB status (r = 0.323; P < 0.01). A histological analysis showed that cagA status was associated with inflammation and atrophy both in the antrum and in the corpus, while homB status was associated with inflammation and atrophy in the corpus. homB gene status might be susceptible to gastric-cancer development such that the homB gene is used as a factor for discriminating the risk of gastric cancer from that of duodenal ulcer.Helicobacter pylori infection is one of the most common infections of mankind and is etiologically associated with gastritis, peptic ulcer disease (PUD), gastric cancer (GC), and gastric mucosa-associated lymphoid tissue lymphoma (19). Most infected people remain asymptomatic. Factors thought to be associated with clinical gastroduodenal diseases include H. pylori virulence, host genetics, and environmental factors, such as diet (11). Putative H. pylori virulence factors associated with an increased risk of a clinical outcome include the cag pathogenicity island, CagA, VacA, BabA, and OipA (5, 9, 22). However, none have been exclusively linked to a specific H. pylori-related disease (e.g., GC).The hom family is a small paralogous family of proteins that contain the C-terminal alternating hydrophobic motif and signal sequences typical of outer-membrane proteins. The homB and homA genes are 90% identical; the differences are confined to the central domain (1). Recent studies suggested that there was a close association between the presence of the homB gene and interleukin-8 secretion from human gastric epithelial cells and that the number of H. pylori isolates binding to gastric cells was related to the number of homB copies present (15). Moreover, the authors proposed that the presence of the homB gene was significantly associated with PUD in Portuguese children and adults less than 40 years of age and that it may be a new H. pylori virulence factor (15, 16). However, there is no study for the association between the homB gene and H. pylori-related diseases in other countries. This study investigated whether there was an association between the homA and homB genes and clinical gastroduodenal diseases and the severity of gastric inflammation in the U.S. and Colombian populations.     

Novel Selective Medium for Isolation of Staphylococcus lugdunensis from Wound Specimens

We compared a novel selective Staphylococcus lugdunensis (SSL) medium with routine media (blood and chocolate agars) for the detection of S. lugdunensis in 990 clinical specimens (from tissue, pus, or wound swabs). Significantly more S. lugdunensis isolates were detected on SSL medium (34/990) than on routine medium (7/990) (P = 0.001, McNemar''s test).     

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.     

Evaluation of a New Selective Medium,BD BBL CHROMagar MRSA II,for Detection of Methicillin-Resistant Staphylococcus aureus in Stool Specimens

We compared the recovery of methicillin-resistant Staphylococcus aureus (MRSA) on a new selective chromogenic agar, BD BBL CHROMagar MRSA II (CMRSAII), to that on traditional culture media with 293 stool specimens. The recovery of MRSA was greater on the CMRSAII agar. Screening of stool samples can identify patients who were previously unknown carriers of MRSA.Active surveillance cultures to identify patients colonized with methicillin-resistant Staphylococcus aureus (MRSA) are recommended as part of an intensive program to control the spread of the organism in health care settings (10, 14). Although the anterior nares are currently the most common site from which to obtain culture specimens to test for MRSA colonization (4, 7, 13, 15), other sites, including the intestinal tract, can be potential sources of unrecognized colonization by this organism (1, 2, 6). Patients with S. aureus (including MRSA) colonization of the intestinal tract are associated with greater contamination of health care workers'' hands and the environment (3). Patients with intestinal colonization by MRSA and concurrent diarrhea contaminate their environment to a significantly greater extent than patients colonized at other body sites (5). Screening stool samples for MRSA can detect patients who are previously unknown carriers and who may not otherwise be cared for using contact precautions, which could potentially lead to increased health care-associated transmission and subsequent MRSA infections (1, 2, 6). It has also been suggested that undetected intestinal colonization by MRSA could be the cause of persistent colonization or recolonization in patients after successful decontamination (8, 11).The use of chromogenic agars has been shown to increase sensitivity and decrease the time to detection of MRSA compared with traditional culture methods (9). However, to date, all currently available chromogenic media used for screening patients for MRSA are recommended only for use with specimens from the anterior nares. We evaluated a new selective and differential chromogenic medium, BD BBL CHROMagar MRSA II (CMRSAII) (BD Diagnostics, Sparks, MD), for its ability to detect MRSA in specimens from multiple body sites, including stool specimens, as part of a large multicenter trial (14a). We report on the evaluation of this medium for detection of MRSA from stool samples collected at our institution.The study was conducted during the period from December 2007 to February 2008 at a 500-bed university-affiliated hospital. A total of 293 stool specimens collected from all wards in the hospital and submitted to the laboratory for Clostridium difficile toxin assay (CDT) were included in the study. Stools were not rejected on the basis of specimen consistency. The specimens were inoculated onto colistin-nalidixic acid (CNA) agar and immediately afterward onto CMRSAII. The plates were incubated in ambient air at 36°C and examined at 24 h (range, 18 to 28 h), and if negative, they were reincubated and examined at 48 h (range, 36 to 52 h). CMRSAII was incubated in the dark to avoid exposure to light. Colonies morphologically consistent with S. aureus recovered on CNA plates were confirmed as S. aureus with a positive coagulase test (Staphaureux, Remel, Lenexa, KS) and confirmed as MRSA by cefoxitin disk diffusion. Mauve-colored colonies present on CMRSAII at 24 and 48 h were presumed to be MRSA and were confirmed as S. aureus with a positive coagulase test. A cefoxitin disk diffusion test was performed if MRSA was not recovered from the CNA plate. Differences in proportions were analyzed using McNemar''s test.MRSA was recovered from 62 (21.2%) of 293 stool specimens. The recovery rate was 60/62 specimens (96.8%) for CMRSAII and 47/62 (75.8%) specimens for CNA media (P = 0.004) (Table ​(Table1).1). Of the 60 MRSA isolates recovered on CMRSAII, 44/60 (73.3%) of MRSA isolates were identified at 24 h and 14/60 (26.7%) at 48 h, whereas the traditional culture method took between 48 to 96 h to confirm an isolate as MRSA. When CMRSAII was compared with cefoxitin disk diffusion, the overall agreement was 97.3% (285/293) at 24 h and 99.3% (291/293) at 48 h. Compared to cefoxitin disk diffusion, CMRSAII had a sensitivity of 84.6% (44/52) at 24 h and 96.8% (60/62) at 48 h and a specificity of 100% at both 24 and 48 h. Traditional culture had a sensitivity of 90.4% (47/52) at 24 h and 75.8% (47/62) at 48 h and a specificity of 100% at both 24 and 48 h compared to the cefoxitin disk diffusion method (Table ​(Table2).2). The sensitivity of the traditional culture method at 48 h was lower than that at 24 h because additional stools were positive on CMRSAII at 48 h and were confirmed by cefoxitin disk diffusion, but none of these were positive at 48 h on traditional media. No false-positive results were identified on CMRSAII after 48 h of incubation.

TABLE 1.

Performance of BD BBL CHROMagar MRSA II compared to colistin-nalidixic acid agar

Evaluation of a New Selective Medium,BD BBL CHROMagar MRSA II,for Detection of Methicillin-Resistant Staphylococcus aureus in Different Specimens

The sensitivity of screening for methicillin-resistant Staphylococcus aureus (MRSA) can be improved by adding other specimen sites to nares. We describe an evaluation of a new selective medium, BBL CHROMagar MRSA II (CMRSAII), for its ability to detect MRSA from different specimen types. CMRSAII is a chromogenic medium which incorporates cefoxitin for the detection of MRSA. A study was performed at four clinical laboratories with the following specimens: 1,446 respiratory, 694 stool, 1,275 skin, and 948 wound specimens and 688 blood culture bottles containing Gram-positive cocci. The recovery of MRSA on traditional culture media was compared to results with CMRSAII. S. aureus was tested by cefoxitin disk diffusion. CMRSAII was interpreted as positive for MRSA at 24 h (range, 18 to 28 h) based solely on the visualization of mauve-colored colonies and at 48 h (range, 36 to 52 h) based on detection of mauve colonies with subsequent confirmation as S. aureus (by coagulase or latex agglutination testing). MRSA was recovered more frequently on CMRSAII (89.8% at 24 h and 95.6% at 48 h) than on traditional culture plates (83.1% at 24 h and 79.8% at 48 h) for all specimen types combined (P < 0.001). The percent sensitivities of CMRSAII at 24- and 48-h reads, respectively, were 85.5 and 92.4% for respiratory specimens, 87.9% and 98.3% for stool specimens, 88.4% and 96.1% for skin specimens, 92.1% and 94.6% for wound specimens, and 100% and 100% for positive blood cultures. The specificity was 99.8% for respiratory specimens and 100% for all others. In conclusion, CMRSAII is a reliable screening medium for multiple specimen types.Controlling the spread of multidrug-resistant microorganisms and especially methicillin-resistant Staphylococcus aureus (MRSA) has become a major infection control objective in the United States (4) and many European countries (3, 4, 21). A part of most programs to control the spread of MRSA is screening of patients (4, 8, 14), and screening has even become mandatory in some countries (11, 31).Traditionally, MRSA screening included mainly the culturing of naris swabs. However, it has been demonstrated that up to 35% of MRSA carriers may be colonized only from sites other than the nares, for example, the throat or the rectum (1, 2, 16).Usage of chromogenic media can improve the sensitivity and pace of MRSA detection (5, 6, 9, 10, 12, 13, 15, 17,19, 20, 22-24, 26-30); however, currently available media that have been marketed at this time are recommended only for nasal specimens.This study was designed to compare the performance of BBL CHROMagar MRSAII (CMRSAII), a chromogenic medium which incorporates cefoxitin, with traditional culture media in the recovery and identification of MRSA isolates from clinical specimens, including respiratory, lower gastrointestinal, and skin specimens as well as wound cultures and blood culture bottles with Gram-positive cocci. In addition, it was designed to determine whether CMRSAII results may be reported as presumptive or definitive with no (or one) confirmatory test at 24 and 48 h of incubation.(These data were presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008.)     

Evaluation of a commercial ELISA for serodiagnosis of Helicobacter pylori infection.

A commercial ELISA for the detection of Helicobacter pylori IgG antibodies was evaluated using serum from 242 patients attending an endoscopy clinic. The efficacy of the ELISA was assessed in relation to the histological detection of H pylori on antral mucosal biopsy specimens. In patients under 61 years of age (n = 138) the ELISA was 97.5% sensitive and 85.5% specific for H pylori infection, with a positive predictive value of 91% and a negative predictive value of 96%. Over the whole group the sensitivity of the ELISA was 93.8% and the specificity 79.3%. The positive predictive value and negative predictive values were, respectively, 90% and 87%. These results suggest that the Bio-Rad GAP IgG H pylori ELISA is suitable for serodiagnosis of H pylori infections for most clinical purposes and thus makes H pylori serology available to routine diagnostic laboratories.     

Detection of Helicobacter pylori in Stool Specimens by PCR and Antigen Enzyme Immunoassay

A highly sensitive seminested PCR assay to detect Helicobacter pylori DNA in feces was developed. PCR with stool specimens and a novel antigen enzyme immunoassay (EIA) for H. pylori detection in feces were evaluated as diagnostic tools and in follow-up with samples from 63 infected and 37 noninfected persons. Infected individuals received eradication therapy followed by endoscopic follow-up 35 days after the start of treatment. At that time, a second stool specimen was obtained from 55 of these patients. Before eradication, the sensitivity of PCR was 93.7% and that of EIA 88.9%. Specificities were 100 and 94.6%, respectively. Of the 55 follow-up specimens, 41 originated from patients from whom H. pylori had been eradicated. Of these, 21 were still positive by PCR and 13 were positive by EIA, indicating that 1 month may be too short a period for follow-up evaluation of stool specimens by these tests.     

Evaluation of liquid media for growth of Helicobacter pylori.

Helicobacter pylori has routinely been isolated and grown on solid media. Recently, we have succeeded in obtaining growth of this organism in several liquid media in large volumes, including tryptic soy broth, Mueller-Hinton broth, brucella broth, brain heart infusion broth, and Columbia broth. Growth was tested in the media with and without supplementation. Growth was obtained after incubation under microaerobic conditions and with CO2 enrichment. Growth in a stationary system versus that in an agitated system was evaluated. Results from these experiments show that H. pylori can be grown in any of the liquid media tested except buffered yeast extract-alpha-ketoglutarate if serum is added. No growth was observed on buffered yeast extract-alpha-ketoglutarate even with serum and other supplementation. Growth of H. pylori in most of the liquid media with supplements was improved if the culture was incubated in a CO2 atmosphere. The findings reported here may be useful in clinical, industrial, and research laboratories that require harvests of large quantities of H. pylori cells.