Diagnosis of Helicobacter pylori in gastric brush and biopsy specimens stained by Rowmanowsky and immunocytochemical methods: comparison with the CLOtest

Helicobacter pylori has been implicated in the pathogenesis of chronic gastritis, gastric and duodenal ulcer, and possibly gastric carcinoma. The organism may be detected by invasive or non‐invasive methods with variable sensitivity. Paired gastric biopsy and gastric brush specimens were collected from 83 patients presenting with non‐ulcer dyspepsia. One biopsy was tested for urease using the CLOtest, the other was processed to paraffin and consecutive sections were stained with haematoxylin and eosin, modified Giemsa and anti‐ H. pylori antisera. The brush specimens were stained with a rapid Romanowsky stain (Hema‐Gurr) and anti‐ H. pylori . The CLOtest was positive in 31 cases, the Giemsa biopsy in 25, the anti‐ H. pylori biopsy in 27, the Hema‐Gurr smear in 27 and the anti‐ H. pylori smear in 19. The sensitivities of the methods after omitting one inadequate biopsy were 96%, 93%, 100%, 96% and 78%, respectively. The specificities were 93% for the CLOtest and 100% for the other methods. While immunocytochemical staining of gastric biopsies may be the most sensitive method for H. pylori identification, the cost and turn around time of the technique may preclude its routine use. Gastric brush cytology is a highly sensitive and specific method for H. pylori detection that is quick and simple to perform. Its application is recommended for the routine diagnosis of H. pylori infection.     

Enzyme immunoassay (EIA) in the rapid diagnosis of gonorrhoea

The diagnostic value of a new, modified enzyme immunoassay (EIA) (Gonozyme; Abbott Laboratories, North Chicago III) was evaluated for the rapid antigenic detection of Neisseria gonorrhoeae in endocervical and urethral specimens. EIA results were compared with those of Gram stain (GS) and conventional culture tests. EIA sensitivity and specificity for male patients attending dermatovenerological clinic were 100% and 96.8% respectively in comparison to 86.7% and 96.8% obtained by Gram staining. For female Obstetrics-Gynaecology patients EIA sensitivity of 100% was highly significant compared to 50% sensitivity by the Gram stain. In culture, 30 strains of N. gonorrhoeae were isolated from 125 male specimens and 2 from 105 specimens from females; this suggests a prevalence of N. gonorrhoeae of 24% in males and 1.9% in females. In vitro antibiotic sensitivity testing indicated 55% resistance to penicillin and 43% to ampicillin in these isolated strains; all were sensitive to erythromycin/tetracycline. 12% of the strains were beta-lactamase producers.     

Culture of Helicobacter pylori: Effect of Preimmersion of Biopsy Forceps in Formalin

Background. Treatment of antibiotic-resistant Helicobacter pylori should be based on bacterial sensitivity testing that requires the ability to isolate the bacterium from gastric mucosal biopsies. The aim of this study was to determine whether the yield for detecting H. pylori infection by culture is reduced by immersion of biopsy forceps in formalin prior to obtaining the specimen.
Materials and Methods. Gastric antral mucosal biopsies (100 specimens) from 50 patients were obtained for culture of H. pylori. An antral biopsy was taken for culture, and with the same forceps a biopsy was taken for histological examination. The biopsy specimen was removed by shaking, whereas the forceps was immersed in 10% buffered formalin for the histological investigation. The forceps was then used without rinsing to obtain a second specimen for culture from an area adjacent to the first site. H. pylori status was determined by histological assessment with the Genta stain and a rapid urease test.
Results. Fifty patients with H. pylori infection documented by histological inquiry and positive rapid urease testing entered the study; 29 had duodenal ulcers, 5 had gastric ulcers, 1 had mucosal associated lymphoid tissue (MALT) lymphoma, and 15 were without ulcer disease. The results of culture both before and after immersion in formalin were identical. One patient had both cultures negative; the sensitivity of culture for detection of H. pylori infection was 98% (95% confidence interval =93%-100%).
Conclusion. Preimmersion of biopsy forceps in formalin does not adversely affect the ability to culture H. pylori.     

Clinical usefulness of urine-based enzyme-linked immunosorbent assay for detection of antibody to Helicobacter pylori: a collaborative study in nine medical institutions in Japan

Background. A urine-based enzyme-linked immunosorbent assay (ELISA) kit for detection of antibody to Helicobacter pylori has been developed in Japan. Urine samples can be obtained noninvasively and are easier and safer to handle than are serum samples. The aim of this study was to examine the clinical usefulness of this urine-based ELISA kit.
Materials and Methods. A pair of random, single-void urine and serum samples was collected from each of 1,061 subjects, including 238 patients with gastroduodenal disease. The sensitivity and specificity of the urine-based ELISA was compared with those of three commercially available serum-based ELISA kits. For those patients with gastroduodenal disease, the urine- and serum-based ELISA results were also compared with those for other diagnostic methods using endoscopic biopsy specimens, such as culture, histology, and rapid urease tests.
Results. Based on the three serum-based ELISA results, the sensitivity, specificity, and accuracy of the urine-based ELISA were 97.7%, 95.6%, and 96.8%, respectively. On the basis of the biopsy test results, the sensitivity (96.2%), specificity (78.9%), and accuracy (91.0%) of the urine-based ELISA were almost equivalent or superior to all three serum-based ELISAs tested. In addition, 10 of the 12 false-positive cases for urine-based ELISA were confirmed to be true positives for antibodies to H. pylori by Western blot analysis and inhibition ELISA.
Conclusions. The urine-based ELISA (URINELISA H. pylori Antibody) is very accurate and should be useful as an alternative to serum-based ELISAs for screening of H. pylori infection.     

Absence of Helicobacter pylori in tonsillar swabs from dyspeptic patients

Aim of this study was to evaluate whether tonsils might be a potential reservoir for Helicobacter pylori (H. pylori) infection. A total of 72 consecutive dyspeptic patients undergoing endoscopy for the first time were studied. For each patient, a bilateral tonsillar swab was performed, before endoscopy, for microbiological culture and immunochemical analysis. Antral biopsies were also collected at endoscopy for microbiological culture, rapid urease test, and histological examination. Helicobacter pylori infection was detected in 42 of 72 (58.3%) patients. All tonsillar specimens were negative for H. pylori on both microbiological culture and immunochemical analysis, suggesting that the tonsils are not an extragastric reservoir for H. pylori infection.     

Specific and sensitive detection of H. pylori in biological specimens by real-time RT-PCR and in situ hybridization

PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.     

Quantitative detection for low levels of Helicobacter pylori infection in experimentally infected mice by real-time PCR

Accurate diagnosis of Helicobacter pylori infection is important in both clinical practice and clinical research. Molecular methods are highly specific and sensitive, and various PCR-based tests have been developed to detect H. pylori in gastric biopsy specimens. We optimized a sensitive and specific quantitative SYBR Green I real-time PCR assay for detection of H. pylori based on amplification of the fragment of a 26-kDa Helicobacter species-specific antigen gene that allows for detection of 5 bacterial cells per PCR sample. Under the assay conditions, SYBR Green I real-time PCR is highly reproducible with a precise log-linear relation in the range of six orders of magnitude of bacterial DNA concentrations. For accurate comparison of H. pylori infection in different tissue samples, the amount of total host DNA in each sample is normalized by TaqMan real-time PCR of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pseudogenes. The developed method was validated in prophilactically immunized and experimentally infected mice and revealed a level of H. pylori gastric colonisation that was below the limit of detection for a rapid urease test. This new method established for a quantitative analysis of H. pylori in the host's stomach may be useful in experimental studies evaluating new anti-H. pylori drugs and vaccines.     

Identification of mixed genotypes in Helicobacter pylori from gastric biopsy tissue by analysis or urease gene polymorphisms

Abstract PCR-RFLP analysis of the urease A and B genes was used to investigate the frequency that Helicobacter pylori from gastric biopsy specimens comprised genotypically distinct strains. Seventy-five strains of H. pylori from 44 subjects from four geographically diverse locations were examined and 31 distinct urease gene profiles were identified. For most cultures, the totals of the RFLP sizes were within 4% of the 2.41-kb PCR product. Five strains were genomically more complex suggesting they contained a mixture of related genotypes. The validity of the test was confirmed by analysis of a known strain mixture and of single colony picks from biopsy cultures. Urease gene PCR-RFLP profiling was shown to be more sensitive than ribotyping for determining the genotypic homogeneity of H. pylori .     

Gastric Juice Polymerase Chain Reaction: An Alternative to Histology in the Diagnosis of Helicobacter pylori Infection

Background. Infection from Helicobacter pylori plays a role in several gastroduodenal diseases. The recent availability of molecular techniques, particularly the polymerase chain reaction (PCR), allows us to detect small amounts of this bacterium. The aims of this study were to compare PCR and histological findings and to ascertain the clinical usefulness of H. pylori PCR identification in different biological samples.
Materials and Methods. We studied 94 consecutive patients. Saliva, gastric juice, and four antral and four body biopsies were obtained from each patients. H. pylori was evaluated histologically in two antral and two body biopsies (Giemsa or Warthin-Starry stain). After extraction, DNA was submitted for PCR amplification using the two primers HPU1 and HPU2, which amplified a 411-bp product from the urease gene A.
Results. Forty-nine patients were H. pylori -positive at histological workup. The sensitivity of PCR was 92% for gastric juice, 73% for antral biopsies, 61% for body biopsies, and 13% for saliva. Of the 45 H. pylori -negative patients at histological assessment, 7 (16%) had positive findings on PCR, mainly when gastric juice was examined.
Conclusions. These results indicate that PCR is as sensitive as histological assessment. We suggest that PCR H. pylori detection in gastric juice is a sensitive method for diagnosing this infection.     

Comparison of two different stool antigen tests for the primary diagnosis of Helicobacter pylori infection in turkish patients with dyspepsia

AIM: To assess the reliability of two different enzyme immunoassays in detecting the Helicobacter pylori status in stool specimens of Turkish patients with dyspepsia. MATERIALS AND METHODS: One hundred and fifty-one patients [74 with nonulcer dyspepsia (NUD), 64 with duodenal ulcer (DU) and 13 with gastric cancer] who were admitted to the endoscopy unit of Istanbul University, Cerrahpasa Medical Faculty for upper gastrointestinal endoscopy because of dyspepsia were enrolled in the study. Helicobacter pylori infection was confirmed in all patients by histology, rapid urease test and culture. A patient was classified as being H. pylori-positive if the culture alone or both the histology and the rapid urease test were positive and as negative only if all of these tests remained negative. Stool samples were obtained from patients to assess the reliability of a monoclonal (FemtoLab H. pylori) and a polyclonal (Premier Platinum HpSA) stool antigen test and to compare the diagnostic accuracies of these two tests. A chi2 test was used for statistical comparisons. RESULTS: Using cut-off values of 0.19 for FemtoLab H. pylori and 0.16 for Premier Platinum HpSA, the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy were 93%, 90%, 98%, 68% and 93% for the monoclonal test and 84%, 67%, 94%, 40% and 81% for the polyclonal test, respectively. The sensitivity, specificity, negative predictive value and diagnostic accuracy of the monoclonal test were significantly greater than those of the polyclonal test (chi2 = 3.98; p < .05 for sensitivity and chi2 = 15.67; p = .000 for specificity, chi2 = 15.78; p = .000 for negative predictive value and chi2 = 6.37; p = .012 for diagnostic accuracy). The bacterial load did not affect the sensitivity of either test. CONCLUSIONS: The monoclonal FemtoLab H pylori test, using a cut-off 0.19, is a very sensitive, specific and easy to perform diagnostic tool for the primary diagnosis of H. pylori infection in Turkish patients with dyspepsia.     

Imprint cytology of core needle biopsy specimens of breast lesions. A rapid approach to detecting malignancies, with comparison of cytologic and histopathologic analyses of 173 cases

OBJECTIVE: To investigate whether imprint cytology of core needle biopsy (CNB) specimens from breast lesions is a useful method of rapidly obtaining additional diagnostic information and potentially can be used to reduce the number of biopsies needed. STUDY DESIGN: Cytologic analysis was performed on 173 breast lesions and compared with their histopathologic diagnoses (143 malignant and 30 benign). For imprint cytology, one CNB specimen was rolled between two slides and stained with Diff-Quik and Papanicolaou stain. RESULTS: The diagnostic overall accuracy of Diff-Quik stain (Papanicolaou stain) was 95.4% (95.9%), with a sensitivity of 96.5% (97.2%), specificity of 90% (90%), positive predictive value of 97.8% (97.8%) and negative predictive value of 84.3% (87.0%). There was no statistically significant difference between the stains. Histopathologic analysis had an overall accuracy of 97.7%, with a sensitivity of 97.2%, specificity and positive predictive value of 100% and a negative predictive value of 88.2%. CONCLUSION: Imprint cytology of CNBs is a sensitive method of detecting malignancies in breast tumors. Diff-Quik is a rapid and reliable approach that can reduce the number of biopsies. Inadequate and suspicious cases should be evaluated based on complementary diagnostic procedures for breast lesions.     

Evaluation of stool antigen test, PCR on ORAL samples and serology for the noninvasive detection of Helicobacter pylori infection in children

BACKGROUND: Endoscopy represents the gold standard for the diagnosis of Helicobacter pylori infection. We evaluated three noninvasive tests in a group of children: the immunoassay for detection of H. pylori stool antigen, the polimerase chain reaction for identification of bacterial DNA on the oral cavity and the serum specific antibodies. MATERIALS AND METHODS: One hundred and ninety children underwent endoscopy for various gastrointestinal symptoms. H. pylori stool antigen and anti-H. pylori antibodies were assayed by commercial kits. The bacterial DNA on saliva and oral plaque was detected by a seminested PCR. RESULTS: Based on the positivity of culture or urease rapid test and histology, infection was detected in 47 patients. The statistical analysis showed that, for the detection of the infection, stool antigen assay is more effective in sensitivity and negative predictive value (91.5% and 96.5%), whereas specificity and positive predictive values appear slightly better in serology (89.6% and 76.0%). Correlations between serum IgG both with patients' age (r = 0.21, p < .05) and H. pylori stool antigen (r = 0.47, p < .01) were found. The search for bacterial DNA on oral samples proved to be very specific (99.1% on saliva and 98.2% on plaque), but insensitive (22.2% and 25.7%). CONCLUSIONS. In children H. pylori stool antigen represents a sensitive test, suitable for detecting H. pylori infection. Serum IgG proved to be more specific; the PCR on the oral cavity resulted as being a very specific, but insensitive test.     

Real-time PCR improves Helicobacter pylori detection in patients with peptic ulcer bleeding

Background and Aims

Histological and rapid urease tests to detect H. pylori in biopsy specimens obtained during peptic ulcer bleeding episodes (PUB) often produce false-negative results. We aimed to examine whether immunohistochemistry and real-time PCR can improve the sensitivity of these biopsies.

Patients and Methods

We selected 52 histology-negative formalin-fixed paraffin-embedded biopsy specimens obtained during PUB episodes. Additional tests showed 10 were true negatives and 42 were false negatives. We also selected 17 histology-positive biopsy specimens obtained during PUB to use as controls. We performed immunohistochemistry staining and real-time PCR for 16S rRNA, ureA, and 23S rRNA for H. pylori genes on all specimens.

Results

All controls were positive for H. pylori on all PCR assays and immunohistochemical staining. Regarding the 52 initially negative biopsies, all PCR tests were significantly more sensitive than immunohistochemical staining (p<0.01). Sensitivity and specificity were 55% and 80% for 16S rRNA PCR, 43% and 90% for ureA PCR, 41% and 80% for 23S rRNA PCR, and 7% and 100% for immunohistochemical staining, respectively. Combined analysis of PCR assays for two genes were significantly more sensitive than ureA or 23S rRNA PCR tests alone (p<0.05) and marginally better than 16S rRNA PCR alone. The best combination was 16S rRNA+ureA, with a sensitivity of 64% and a specificity of 80%.

Conclusions

Real-time PCR improves the detection of H. pylori infection in histology-negative formalin-fixed paraffin-embedded biopsy samples obtained during PUB episodes. The low reported prevalence of H. pylori in PUB may be due to the failure of conventional tests to detect infection.     

Realities of diagnosing Helicobacter pylori infection in clinical practice: a case for non-invasive indirect methodologies.

BACKGROUND: The current, arbitrarily defined gold standard for the diagnosis of H. pylori infection requires histologic examination of two specially stained antral biopsy specimens. However, routine histology is potentially limited in general clinical practice by both sampling and observer error. The current study was designed to examine the diagnostic performance of invasive and non-invasive H. pylori detection methods that would likely be available in general clinical practice. METHODS: The diagnostic performance of rotating clinical pathology faculty using thiazine staining was compared with that of an expert gastrointestinal pathologist in 38 patients. In situ hybridization stains of adjacent biopsy cuts were also examined by the expert pathologist for further comparison. Receiver operator characteristic (ROC) analysis was performed to evaluate whether the diagnostic performance of the expert pathologist differed depending upon the histologic method employed. A similar analysis was made to evaluate the diagnostic performance of pathology trainees relative to the expert. In the absence of an established invasive gold standard, non-invasive testing methods (rapid serum antibodies, formal Elisa antibodies and carbon-14 urea breath testing) were evaluated in 74 patients by comparison with a gold standard defined using a combination of diagnostic tests. RESULTS: Using either rapid urease testing of biopsy specimens or urea breath testing as the gold standard for comparison, the diagnostic performance of the rotating clinical pathology faculty was inferior to that of the expert gastrointestinal pathologist especially with regard to specificity (e.g., 69 percent for the former versus 88 percent, with the latter relative to rapid urease testing). Although interpretation of in situ hybridization staining by the expert appeared to have an even higher specificity, ROC analysis failed to show a difference. The mean ROC areas for thiazine and in situ hybridization staining for trainee pathologists relative to the expert were 0.88 and 0.94, respectively. In untreated patients, urea breath testing had a sensitivity and specificity of 100 percent as compared with thiazine staining with a sensitivity of 83 percent and a specificity of 97 percent. Post-therapy, breath testing had a sensitivity of 100 percent but a specificity of only 86 percent as compared with invasive testing with a sensitivity and specificity of 100 percent. Rapid serum antibody testing and formal Elisa antibody testing agreed in 93 percent of cases (Kappa 0.78) with the rapid test being correct in three of the four disagreements. CONCLUSIONS: The current study illustrates a number of realities regarding H. pylori diagnosis. There is no diagnostic gold standard in general clinical practice. Accurate interpretation of specially stained slides is a learned activity with a tendency towards overdiagnosis early on. Urea breath testing is likely to be the diagnostic method of choice for untreated patients in general clinical practice although antibody testing is almost as accurate. Rapid antibody tests are at least as accurate as formal Elisa antibody tests. Urea breath testing is useful for confirming cure after therapy, but false-positive results may occur in some patients.     

Evaluation of non-culture diagnosis of invasive meningococcal disease by polymerase chain reaction (PCR)

Antibiotic treatment prior to transport or admission to hospital has reduced the proportion of cases of invasive meningococcal disease (IMD) from which Neisseria meningitidis can be isolated by standard microbiological techniques. Identification of meningococci by polymerase chain reaction (PCR) was assessed in relation to microbiological diagnosis for cases over a 4-year period between 1998 and 2001. A screening assay for the IS1106 gene was used to detect meningococcal DNA and five additional assays for siaD and orf-2 genes were performed to determine the serogroup. PCR results were compared with results of bacteriological culture, other laboratory test results and clinical data. The sensitivity of the PCR assay for culture-confirmed cases was 98.5%. The specificity of the assay was 96% based on test results for patients from whom other bacteria were isolated, children with viral meningitis and afebrile negative controls. The siaD B/C/W-135 and Y as well as the orf-2 gene for serogroup A PCR assays were able to determine the serogroup for 75.2% of cases that were positive by PCR screening assay. When isolates from patients with IMD were tested by both agglutination and PCR, the results agreed in all cases. PCR is a useful tool for diagnosis of IMD when Gram stain and culture tests are negative due to antibiotic treatment prior to collection of samples for microbiological analyses.     

Validation of Diagnostic Tests for Helicobacter pylori with Regard to Grade of Atrophic Gastritis and/or Intestinal Metaplasia

Background and Aims:  To evaluate the validity of the biopsy-based tests (histology, culture, and urease test) and serology in detecting current Helicobacter pylori infection against a background of atrophic gastritis (AG) or intestinal metaplasia (IM).
Methods:  Helicobacter pylori infection was diagnosed in 651 subjects, using the predefined gold standard for H. pylori tests. The sensitivity, specificity, and positive and negative predictive values of culture, CLOtest, histology (Giemsa stain), and serology were calculated with regard to the histological grade of AG and IM. The level of serum pepsinogen (PG) I and II was also measured as a marker for the presence of AG.
Results:  In the study population (n = 651), sensitivity and specificity, respectively, were as follows: culture, 56.2 and 100%; histology, 93.0 and 94.0%; CLOtest, 80.4 and 96.7%; serology, 96.0 and 67.5%. If the analysis is limited to those without AG or IM (n = 158) or to those younger than 40 years (n = 69), all tests, except for culture, had a sensitivity and specificity >90%. The sensitivity of CLOtest and the specificity of serology markedly decreased with progression of AG and IM, and serology was less specific in the presence of AG, as determined by a PG I/II ratio ≤4.1 (specificity, 83.7% vs 40.7% in PG I/II >4.1 and ≤4.1, respectively).
Conclusions:  Any one of biopsy-based tests or serology was found to be excellent for identifying current H. pylori infection among individuals without AG or IM and/or younger patients (<40 years). However, a combination of at least two tests is necessary in the clinical setting of AG or IM.     

Primary screening with liquid-based cytology in an unscreened population in rural China,with an emphasis on reprocessing unsatisfactory samples

OBJECTIVE: To determine the sensitivity and specificity of liquid-based cytology in a high-risk, unscreened population. STUDY DESIGN: A cross-sectional comparative trial of multiple screening techniques. This report focuses on the performance of liquid-based cytology using biopsy as the standard. RESULTS: In total, 1,997 women were screened. All subjects had a minimum of five cervical biopsies. Of the subjects, 4.3% had CIN 2 on biopsy. The ThinPrep Pap with ASCUS as positive had a sensitivity for CIN 2 of 94% and specificity of 78%; the sensitivity for CIN 3 was 98% and for cancer, 100%. The unsatisfactory rate due to bloody specimens was 7.9% (158/1,997). After reprocessing, the unsatisfactory rate was 0.15% (3/1,997). CONCLUSION: Reprocessing effectively recovers ThinPrep samples otherwise unsatisfactory due to blood and mucus. The ThinPrep Pap test is a highly sensitive screening test when used in a high-risk, unscreened population.     

LightCycler real-time PCR for rapid detection and quantitation of Mycobacterium leprae in skin specimens

Diagnosis of leprosy is usually based on clinical features and skin smear results including the number of skin lesions. Mycobacterium leprae is not cultivable and bacterial enumeration by microscopic examination is required for leprosy classification, choice in choosing and monitoring chemotherapy regimens, and diagnosis of relapse. However, detection and quantification using standard microscopy yields results of limited specificity and sensitivity. We describe an extremely sensitive and specific assay for the detection and quantification of M. leprae in skin biopsy specimens. Primers that amplified a specific 171-bp fragment of M. leprae 16S rRNA gene were chosen and specificity was verified by amplicon melting temperature. The method is sensitive enough to detect as low as 20 fg of M. leprae DNA, equivalent to four bacilli. The assay showed 100% concordance with clinical diagnosis in cases of multibacillary patients, and 50% of paucibacillary leprosy. The entire procedure of DNA extraction and PCR could be performed in c. 3 h. According to normalized quantitative real-time PCR, the patients in this study had bacilli numbers in the range of 1.07 x 10(2) -1.65 x 10(8) per 6-mm3 skin biopsy specimen. This simple real-time PCR assay is a facile tool with possible applications for rapid detection and simultaneous quantification of leprosy bacilli in clinical samples.     

Diagnosis of Helicobacter pylori infection and determination of clarithromycin resistance by fluorescence in situ hybridization from formalin-fixed, paraffin-embedded gastric biopsy specimens

A reliable diagnostic test for Helicobacter pylori is important in clinical practice and research. The ideal diagnostic test for H. pylori should be sensitive, specific, and cost-effective. Helicobacter pylori resistance to clarithromycin is a common reason for failure of eradication therapy. The aim of this study was to evaluate the fluorescent in situ hybridization (FISH) method to detect H. pylori and determine clarithromycin resistance in formalin-fixed, paraffin-embedded gastric biopsy specimens. One hundred seventeen gastric biopsy specimens from patients with dyspepsia were examined for the presence of H. pylori by conventional culture, FISH, and histopathological methods. A set of fluorescent-labeled oligonucleotide probes binding to either H. pylori 16S rRNA or 23S rRNA sequences were used for FISH analysis. Phenotypic antibiotic susceptibilities of the isolates were tested using the Epsilometer test method (E test). Helicobacter pylori was detected in 70 of 117 biopsy specimens by histopathological examination and FISH, whereas it was detected in 47 specimens by culturing. Histopathology and FISH techniques failed to identify H. pylori in 1 biopsy sample isolated by culture. Clarithromycin resistance was found in 11 of 46 H. pylori isolates using the E test method. All of the phenotypic resistance measurements of isolates were correlated with genotypic clarithromycin resistance. Eleven clarithromycin-resistant strains were identified by FISH. The diagnosis of H. pylori infection and the determination of clarithromycin resistance in formalin-fixed, paraffin-embedded specimens using FISH is promising because it provides a rapid, reliable, and culture-independent diagnosis.     

The Best Gastric Site for Obtaining a Positive Rapid Urease Test

Background Rapid urease tests (RUTs) provide a simple, sensitive method of detecting Helicobacter pylori infection.
Objectives. Our aim, therefore, was to determine whether the yield of detecting H. pylori infection by RUT varied depending on the site of gastric biopsy.
Materials and Methods. Gastric biopsies were obtained from 50 patients for RUT by use of hp fast (GI Supply, Camp Hill, PA). Biopsies were taken from the prepyloric greater curve antrum, from the gastric angle, and from the greater curve in mid-corpus. One biopsy specimen was placed in the RUT gel, and the biopsy from the adjacent mucosa was placed in formalin for subsequent histological evaluation by using the Genta stain. RUTs were examined and scored at intervals of 5, 10, 15, 30, and 45 minutes and after 1, 2, 4, and 24 hours.
Results. Fifty patients were entered in the test (150 RUTs), 32 having H. pylori infection. There were no false-positive RUTs (specificity, 100%). The gastric angle site was positive in 100%. The prepyloric site was positive in 87%, and the corpus site was positive in 84.4% ( p < .052 for angle or prepyloric antrum versus corpus). The most common pattern was for all to be positive (74%). The median time to positivity was similar with angle and prepyloric sites (37.5 and 60 minutes, respectively, p = not significant) and shorter than the corpus biopsy (180 minutes); ( p < .05 for angle or prepyloric antrum versus corpus).
Conclusion. The maximum probability for detecting H. pylori infection using a RUT is to obtain a biopsy from the gastric angle. To prevent missing a positive result when intestinal metaplasia is present, we recommend that (at a minimum) biopsies be taken from both the angle and the corpus.