RIDASCREEN® FemtoLab H. pylori

Article no: C2301

R-Biopharm AG, An der neuen Bergstraße 17, D-64297 Darmstadt, Germany Phone: +49 (0) 61 51 81 02-0 / Fax: +49 (0) 61 51 81 02-20 )

1. Intended use

For in vitro diagnostic use. The RIDASCREEN® FemtoLab H. pylori test is an immunoassay for the qualitative determination of antigens in stool samples.

2. Summary and explanation of the test

In 1984, Marshall and Warren found a campylobacter-like organism in the mucosa of the antrum and the corpus in patients with and peptic ulcers of the by using histology. Today, the causal relationship of Helicobacter pylori at the onset of gastrointestinal illnesses is now recognised. with H. pylori lead to inflammations which have a causal relationship with chronic gastritis, ulcers in the and , and stomach cancers. This is confirmed by the, in most cases, successful healing of gastritis and ulcers after eradication therapy. H. pylori has developed various protection mechanisms in order to survive in the acidic, bactericidal environment of the stomach. The enzyme, , splits into ammonia and and neutralises the gastric acid. The production of catalase and superoxidedismutase protects against attacks from neutrophiles in the gastric mucosa. Many H. pylori positive patients develop gastritis and approx. 10 % of the patients develop ulcers. 90 % of patients with small intestine or stomach ulcers are H. pylori positive, whatever their age. The reasons for this are currently the subject of research all over the world, as are the transmission routes of the organism. There are two basic approaches to the diagnosis of H. pylori infections: the direct approach of determining the organism and the indirect approach of determining the antibodies against H. pylori which have been produced by the patient. Among the direct, but invasive methods of determining an are the urease- quick test, histology or cultivation of the organism from material. Culturing H. pylori from biopsy material is difficult and time consuming. The technical difficulties can lead to false negative results, i.e. low sensitivity. In addition, H. pylori is inclined to colonise the gastric mucosa in islands and can therefore be overlooked during endoscopic examinations. Another direct method of determining H. pylori is the breath test. In this case, the are determined from the carbon dioxide produced by the bacterial enzyme urease. The breath test is very sensitive and specific but requires special measuring instruments and the patient has to take isotope-labelled urea. One method which is frequently used is the serological determination of H. pylori specific antibodies. This is an indirect method where the antibodies produced by the patient are detected against H. pylori. The sensitivity and specificity vary greatly between the tests from different manufacturers. Furthermore, monitoring the success of an eradication therapy using serological methods is unsatisfactory since the antibody titer only drops slowly over a period of months. The ® RIDASCREEN FemtoLab H. pylori test is an enzyme immunoassay (EIA) in microwell-plate format for the direct, non-invasive determination of H. pylori antigens in human stools. The test is based on monoclonal antibodies, fluctuations between the individual lots being avoided. By determining the antigens directly, it is possible to support both the position of an initial diagnosis and to check the

RIDASCREEN® FemtoLab H. pylori 10-08-10 2 success of the eradication therapy 4 to 6 weeks after its completion or detect the recurrence of an infection.

3. Test principle

® The RIDASCREEN FemtoLab H. pylori test uses an enzyme immunoassay with dual amplification technology for determining H. pylori antigens in stool samples. The wells in the microwell plate are coated with monoclonal antibodies against H. pylori antigens. The supernatant of a stool suspension and the peroxidase-labelled monoclonal antibodies (enzyme conjugate) are pipetted into the wells. During the incubation, H. pylori antigens from the stool suspension attach themselves both to the antibodies on the test plate and the peroxidase-labelled antibodies, thus forming a 'sandwich complex'. Enzyme conjugate which remains unattached is removed by washing the test plate. After adding the substrate, the attached enzyme changes the colour of the previously colourless solution in the wells of the microwell plate to blue if the test is positive. On adding the stop reagent, the colour changes from blue to yellow. The extinction is proportional to the concentration of H. pylori antigens present in the sample.

4. Reagents provided

There are enough reagents in the pack for 96 determinations. Plate 96 det. Microwell plate coated with antibodies; 12 dividable microwell strips with 8 wells each; coated with monoclonal antibodies against H. pylori antigens; in sealable aluminium bag with desiccant; 1 piece cover film for microwell strips; 100 pieces sampling device (wooden splint) Diluent | 2 100 ml Sample dilution buffer; 75 mM phosphate buffer; pH 7.4; with antimicrobial additives; ready for use; green coloured Wash 100 ml Wash buffer concentrate (x10); 10 x concentrated 250 mM phosphate buffer; pH 7.4; with detergent and antimicrobial additives

Control ⎮+ 2 ml Positive control; inactivated, fractionised H. pylori lysate in 75 mM phosphate buffer; pH 7.4; with antimicrobial additives; ready for use; red coloured

Control ⎮- 2 ml Negative control; 75 mM phosphate buffer; pH 7.4; with antimicrobial additives; ready for use; blue coloured Conjugate 7 ml Enzyme conjugate; monoclonal antibodies against H. pylori antigens; peroxidase conjugate in 75 mM phosphate buffer; pH 7.4; with antimicrobial additives; ready for use; green coloured

RIDASCREEN® FemtoLab H. pylori 10-08-10 3

Substrate 12 ml Urea peroxide/TMB; ready for use Stop 12 ml Stop solution; 1 N sulphuric acid; ready for use

5. Storage instructions

All reagents must be stored at 2 – 8 °C and can be used until the date printed on the label. The diluted wash buffer can be used for up to three months, providing it is stored between 2 and 8 °C. Microbial contamination must be prevented. After the expiry date, the quality guarantee is no longer valid. The aluminium bag must be opened with scissors in such a way that the clip seal is not torn off. Any microwell strips which are not required must be placed in the aluminium bag immediately and stored at 2 – 8 °C. The colourless substrate must also be protected from direct light to prevent it from decomposing or turning blue due to auto-oxidation. Once the substrate has turned blue, it must not be used.

6. Additional necessary reagents – and necessary equipment

6.1. Reagents

- Distilled or deionised water

6.2. Accessories

- Test tubes

- Washing unit for mictrotitration pipettes or multichannel pipettes (300 µl)

- Photometer for microwell plates (450 nm and reference filter ≥ 600 nm where necessary)

- Centrifuge (at least 5000 rpm)

- Shaker, suitable for microwell plates

- Vortex mixer

- Stop clock

- Measuring cylinder (1000 ml)

- Micropipette for 50 - 100 µl and 1 ml volume

- Filter paper (laboratory towels)

- Waste container containing 0.5 % hypochlorite solution

7. Precautions for users

For in vitro diagnostic use only.

RIDASCREEN® FemtoLab H. pylori 10-08-10 4 This test must only be carried out by trained laboratory personnel. The guidelines for working in medical laboratories must be followed and the instructions for carrying out the test must be strictly adhered to.

The positive controls in the kit contain inactivated helicobacter antigens. In spite of this, they and the patient samples must be treated as potentially infectious and handled in accordance with the national safety regulations.

Samples or reagents must not be pipetted by mouth and contact with injured skin or mucous membranes must be prevented.

When handling the samples, wear disposable gloves and when the test is finished, wash your hands. Do not smoke, eat or drink in areas where samples or test reagents are being used.

Sample dilution buffer, wash buffer, positive and negative control and conjugate contain antimicrobial additives. This substance must not be allowed to come into contact with the skin or mucous membrane.

Hydrogen peroxide can cause burns. Handle with care.

The stop reagent contains 1 N sulphuric acid. Avoid contact with the skin and clothing. If the skin is contaminated with the stop reagent, rinse it off with water.

All reagents and materials which have come into contact with potentially infectious samples must be treated with suitable disinfectants or autoclaved at 121 °C for at least 1 hour. CAUTION: To prevent the formation of poisonous gases, any liquid waste containing stop reagent must be neutralised before adding to hypochlorite solution.

8. Specimen collection and storage

The test material must be stored at 2 – 8 °C until it is used in the test. If the material is not going to be used in the test within 3 days, we recommend storing it at –20 °C or colder. Multiple freezing and thawing of the sample must be avoided. Stool samples or rectal swabs should not be collected in transport containers which contain transport media with preservatives, animal sera, metal ions, oxidising agents or detergents since these may interfere with the RIDASCREEN® FemtoLab H. pylori test. If rectal swabs have to be used, make sure that sufficient stool material (approx. 200 mg) is collected to carry out the test.

9. Test procedure

9.1. General information

RIDASCREEN® FemtoLab H. pylori 10-08-10 5 All reagents and the microwell plate Plate must be brought to room temperature (20 – 25 °C) before use. The microwell strips must not be removed from the aluminium bag until they have reached room temperature. The reagents must be thoroughly mixed immediately before use. After use, the microwell strips (in sealed bags) and the reagents must be stored at 2 – 8 °C. Once used, the microwell strips must not be used again.

The reagents and microwell strips must not be used if the packaging is damaged or the vials are leaking. In order to prevent cross contamination, the samples must be prevented from coming into direct contact with the kit components. The test must not be carried out in direct sunlight. We recommend covering the microwell plate or placing film on it to prevent evaporation losses.

9.2. Preparing the wash buffer

Mix 1 part wash buffer concentrate Wash with 9 parts distilled water. Any crystals present in the concentrate must be dissolved beforehand by warming in a water bath at 37 °C.

9.3. Preparing the samples

Place 1 ml RIDASCREEN® sample dilution buffer Diluent | 2 in a labelled test tube. Mix the stool sample and add a pea-sized quantity (approx. 200 mg) of the patient’s stool to the sample buffer using the wooden splint provided. With liquid stool samples, use 200 µl of the stool. After making a suspension using the vortex mixer (15 seconds) centrifuge the sample down for 5 minutes at 5000 rpm. Use the supernatant in the test. A new wooden splint must be used for each sample. The stool suspension can be kept for 24 hours at 2 – 8 °C but it must not be frozen.

9.4. First incubation

After selecting a sufficient number of wells in the frame, add 50 µl of the supernatant from the stool sample, 50 µl positive control Control + or 50 µl negative control Control | - to each well. After this, pipette 50 µl of the enzyme conjugate Conjugate directly into the sample or controls. Then incubate the plate at room temperature (20 – 25 °C) on a shaker for 60 ±5 minutes. Cover the wells with the film provided cut to the appropriate size.

9.5. Washing

Careful washing is important in order to achieve the correct results and should therefore take place strictly according to the instructions. The incubated substance in the wells must be emptied into a waste container containing hypochlorite for disinfection. After this, knock out the plate onto absorbent paper in order to remove the residual moisture. Then wash the plate 5 times using 300 µl wash buffer each time. Make sure that the wells are emptied completely by knocking them out after each wash on a part of the absorbent paper which is still dry and unused.

When using an automated washer, make sure that the machine is correctly adjusted to the type of microwell plate being used. Furthermore, a stool suspension which is not completely

RIDASCREEN® FemtoLab H. pylori 10-08-10 6 particle-free before the first wash should be removed manually by centrifuging it from the cavities in order to avoid blocking the wash needles. Also make sure that all of the liquid is sucked away during each washing phase. After washing for the last time, knock out the plate thoroughly onto clean absorbent paper or a laboratory towel in order to remove any residual moisture.

In order to achieve the optimum washing results, it is advisable to wash in overflow mode with at least 600 μl wash buffer per well and wash step. The number of wash steps can be increased to more than 5 and this can occasionally lead to better washing results.

9.6. Second incubation

Add 100 µl Substrate solution Substrate to each well. Then incubate the plate at room temperature (20 – 25 °C) for 10 minutes in the dark. After this, stop the enzyme reaction by adding 100 µl stop solution Stop to each well. After mixing carefully (by lightly tapping the side of the plate) measure the extinction at 450 nm using a reference wavelength ≥ 600 nm (optional). Then calibrate the zero against air i.e. without a microwell plate.

Note : High positive samples may cause the substrate to precipitate as a black sediment.

10. Quality control – indications of reagent expiry

For quality control purposes, positive and negative controls must be used each time the test is carried out in order to make sure that the test has been carried out correctly and the reagents are stable. The test has been carried out correctly if the extinction (O.D.) for the negative control at 450 nm is less than 0.14 (OD450 / 620 to 650 nm < 0.10) and the measured value for the positive control at 450 nm is greater than 1.04 (OD450 / 620 to 650 nm >1.00). If the value is greater than 0.14 for monochromatic or 0.10 for bichromatic measurement, this may indicate that there was insufficient washing. If the values differ from those required or if the substrate is turbid or has turned blue before adding to the wells, this may be an indication that the reagents have expired. If the stipulated values are not met, the following points must be checked before repeating the test:

- Expiry date of the reagents used

- Functionality of the equipment being used (e.g. calibration)

- Correct test procedure

- Visual inspection of the kit components for contamination or leaks – a substrate solution which has turned blue must not be used.

If the conditions are still not fulfilled after repeating the test, please consult the manufacturer or your local R-Biopharm distributor.

11. Evaluation and interpretation

RIDASCREEN® FemtoLab H. pylori 10-08-10 7 11.1. Calculating the cut-off

Measurement at 450 nm against a reference wavelength between 620 and 650 nm. The cut-off is established as follows: Cut-off = 0.150 OD

If it is not possible to measure against a reference wavelength on the photometer being used, the cut-off at 450 nm is as follows: Cut-off = 0.190 OD

11.2. Test result

Samples are considered positive if their extinction is more than 0.02 units above the calculated cut-off.

Samples are considered equivocal and must be repeated if their extinction is within the range 0.02 units above or below the cut-off. If repeating the test with a fresh stool sample again yields a value in the grey range, the sample must be considered negative.

Samples with extinctions more than 0.02 units below the calculated cut-off must be considered negative.

12. Limitations of the method

The RIDASCREEN® FemtoLab H. pylori test determines a highly specific antigen of Helicobacter pylori in stool samples. The test cannot be used to derive a relationship between the determined extinction and the occurrence of serious clinical symptoms. The results obtained must always be interpreted in combination with the clinical picture.

A positive result does not rule out the presence of another infectious pathogen.

A negative result does not necessarily mean that there is no Helicobacter pylori infection. This can be caused by intermittent excretion of the pathogen or by the quantity of antigens in the sample being too small. If the patient is anaemic or has suspected H. pylori infection, another stool sample should be tested after four weeks.

An equivocal result may be caused by the distribution of the antigen in the stool sample being non- homogeneous. If this happens, a second suspension should be tested from the same sample or a second stool sample collected for the test.

Epidemiological studies show that infections with H. pylori are widespread throughout the whole world. 25 – 50 % of the population in Europe and North America are infected. Studies in Asia, Africa and Latin America yielded prevalences of 70 – 90 % and even higher. The infection rate of H. pylori

RIDASCREEN® FemtoLab H. pylori 10-08-10 8 relates to age, the ethnical origin and standard of living. In the United States for example, the infection prevalence increases with each year of life by around 1 %. The expected values are dependent on the geographical location and the kind of patient population examined. The proportion of positive test results can vary depending on the method of determination and the method of sample collection and handling used.

The RIDASCREEN® FemtoLab H. pylori test is a qualitative test and does not provide quantitative information. Test results should be interpreted in relation to clinical data and/or other test procedures. Antibiotics, proton pump inhibitors and bismuth preparations suppress the growth of H. pylori. Stool sample testing and later eradication control may be carried out after 2 weeks at the earliest after discontinuing proton pump inhibitors or bismuth preparations or 4 weeks after discontinuing antibiotics. A positive test result alone is not enough to indicate eradication therapy; other methods for confirming H. pylori infection may be necessary. However, in order to determine ulcers, autoimmune gastritis or tumours, a differential diagnosis is necessary using an invasive endoscopic method.

13. Performance characteristics

13.1. Test quality

Study 1: Primary diagnosis for adult patients

The RIDASCREEN® FemtoLab H. pylori test was evaluated on 356 patients (201 female and 155 male, aged 18 - 82 years) who were being examined by at 10 German clinics because of abdominal pain and dyspepsia. The test procedure was carried out on blind stool samples in independent laboratories. The patients showed different pathological findings: mild gastritis (n = 61), C gastritis (n = 98), B gastritis (n = 144), erosions of the antrum (n = 11), A gastritis (n = 2), gastric ulcer (n = 5), duodenal ulcer (n = 3), glandular carcinoma (n = 2), submucosal tumour (n = 1), Schatzki's ring (n = 1), Crohn gastritis (n = 1) and asymptomatic (n = 27). The results of the RIDASCREEN® FemtoLab H. pylori test were compared with the histological findings. A diagnostic sensitivity of 95.3 % and a diagnostic specificity of 97.1 % was established for the RIDASCREEN® FemtoLab H. pylori test. The confidence intervals (CI) were calculated using the accurate binomial method. The results are listed in Table 1.

Table 1: Primary diagnosis for adult patients using the RIDASCREEN® FemtoLab H. pylori test and the histological reference test

RIDASCREEN® FemtoLab H. pylori 10-08-10 9

RIDASCREEN® Histology FemtoLab H. pylori + - + 141 6 - 7 202

Sensitivity: 95.3 % Specificity: 97.1 % ± 95 % CI 90.5 - 98.1 % ± 95 % CI 93.8 - 98.9 %

Study 2: Primary diagnosis for paediatric patients

The RIDASCREEN® FemtoLab H. pylori test was tested in a study with stool samples from children who were undergoing endoscopic examination because of abdominal pain and/or other intestinal complaints. 239 children (124 male, 115 female; aged 6 months - 18 years) were examined from 3 paediatric gastroenterology clinics in Europe. The histology and the culture test were used as a reference. Patients were H. pylori positive if the histology and/or culture test showed positive and H. pylori negative if both tests were negative. The RIDASCREEN® FemtoLab H. pylori test showed a sensitivity of 98.6 % and a specificity of 99.4 %. The confidence intervals (CI) were calculated according to the accurate binomial method.

Table 2: Primary diagnosis for paediatric patients using the RIDASCREEN® FemtoLab H. pylori test and the histological/culture reference test

Histology - culture RIDASCREEN® FemtoLab H. pylori + - + 70 1 - 1 167

Sensitivity: 98.6 % Specificity: 99.4 % ± 95 % CI 92.4 – 100 % ± 95 % CI 96.7 – 100 %

Study 3: Eradication monitoring

40 H. pylori infected children with abdominal pain (age 3 to 15 years) from two gastroenterological clinics were included in this study. H. pylori infection was found by the breath test and serology. All 40 stool samples were identified by the RIDASCREEN® FemtoLab H. pylori test as H. pylori positive (sensitivity 100 %; 95 % CI = 91.2 – 100 %). Eradication was carried out using triple-therapy over 7 days. Eradication was checked 4 weeks after the end of the therapy by means of breath tests. The RIDASCREEN® FemtoLab H. pylori test showed a sensitivity of 100 % and a specificity of

RIDASCREEN® FemtoLab H. pylori 10-08-10 10 98.9% in comparison to the results of the breath test. The confidence intervals were determined according to the accurate binomial method.

Table 3: Eradication monitoring with the RIDASCREEN® FemtoLab H. pylori test and the breath test 4 week after the conclusion of the eradication therapy

® Breath test RIDASCREEN FemtoLab H. pylori + -

+ 8 1 - 0 31 Sensitivity: 100 % Specificity: 96.9 % ± 95 % CI 63.1 - 100 % ± 95 % CI 83.8 - 99.9 %

13.2. Cross reactivity

The RIDASCREEN® FemtoLab H. pylori test is highly specific for H. pylori antigens. No cross reactivity was found for any of the micro-organisms listed below (Table 4). In contrast to this, H. pylori gave a positive test result. Table 4: Cross reactions with pathogenic micro-organisms

Acinetobacter lwoffii 1 Proteus mirabilis 1, 2 Aeromonas hydrophila anaerogenes 1 Proteus vulgaris 1,2 Aeromonas hydrophila hydrophila 1 Providencia stuartii 1 Bacteroides thetaiotaomicron 2 Pseudomonas aeruginosa 1 Bacteroides vulgatus 2 Pseudomonas fluorescens 1 Campylobacter fetus 1, 2 Pseudomonas putida 1 Campylobacter jejuni 2 Salmonella agona 1 Candida albicans 2 Salmonella choleraesuis 1 Citrobacter freundii 1, 2 Salmonella infantis 1 Enterobacter cloacae 1 Salmonella ohio 1 Enterococcus faecalis 1, 2 Salmonella typhimurium 1 Enterococcus faecium 1 Serratia proteamaculans 1 Escherichia coli 1, 2 Shigella flexneri 1 Escherichia hermannii 1 Shigella sonnei 1 Eubacterium aerofaciens 2 Staphylococcus aureus 1 Klebsiella pneumoniae 2 Streptococcus agalactiae 1

RIDASCREEN® FemtoLab H. pylori 10-08-10 11 Lactoccocus lactis 1 Streptococcus dysgalactiae 1 Listeria innocua 1 Yersinia enterocolitica 2 Peptostreptococcus productus 2

1 Each strain was tested at a concentration of > 1×108 organisms/ml sample dilution buffer. 2 Each strain was tested in the RIDASCREEN® FemtoLab H. pylori test at a concentration of 100 µg lysat protein/ml sample dilution buffer.

13.3. Detection limit

The RIDASCREEN® FemtoLab H. pylori test can detect 5 femtomol antigens per gram stool.

13.4. Precision

Intra- and inter- assay variances were determined by testing negative (n = 2), weakly positive (n = 2), medium positive (n = 2) and strongly positive samples (n = 2). The measurements were carried out in 3 independent laboratories in Europe. Each sample was measured ten times in each of these laboratories. The intra- and inter-assay coefficient of variation (CV) were calculated and are shown in Table 5. The table shows the lowest and highest OD values and intra- and inter-assay variances for the different stool samples in each case.

Table 5: Intra- and inter-assay variances for the RIDASCREEN® FemtoLab H. pylori test

® Weakly Medium Strongly RIDASCREEN Negative FemtoLab H. pylori positive positive positive

OD 450/630 nm 0.02 - 0.03 0.49 - 0.90 1.31 - 2.66 3.00 - 3.77

Intra-assay CV 6.6 - 17.4% 2.7 - 10.1% 2.1 - 4.0% 1.1 - 6.1%

Inter-assay CV 6.0 - 27.1% 27.3 - 31.3% 29.0 - 30.2% 7.5 - 7.8%

RIDASCREEN® FemtoLab H. pylori 10-08-10 12 References 1. Marshall, B.J., Warren, J.R.. 1984. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1(8390): 1311-1314. 2. Dunn, B.E., Cohen, H., Blaser, M.J. 1997. Helicobacter pylori. Clin Microbiol Rev 10: 720- 741. 3. D’Elios, M.M., Andersen, L.P., Del Prete, G. 1998. Inflammation and host response. Curr Opin in Gastroenterology, 14 (suppl. 1): 15-19. 4. Delchier, J.-C., Ebert, M., Malfertheiner, P. 1998. Helicobacter pylori in gastric lymphoma and carcinoma. Curr Opin in Gastroenterology, 14 (suppl. 1): 41-45 5. Feldman, R.A., Eccersley, A.J.P., Hardie, J.M. 1997. Transmisssion of Helicobacter pylori. Curr Op in Gastroenterology 8: 8-12. 6. Graham, D.Y., Klein, P.D., Evans, Jr., D.J., Evans, D.G., Alpert, L.C., Opekun, A.R., Boutton, T.W. 1987. Campylobacter pylori detected noninvasively by the 13C-urea breath test. Lancet 1(8543): 1174-1177. 7. Graham, D.Y., Klein, P.D., Opekun, A.R., Boutton, T.W. 1988. Effect of age on the frequency of active Campylobacter pylori infection diagnosed by the [13C] Urea breath test in normal subjects and patients with . J Infect Dis 157: 777-780. 8. Barthel, J.S., Everett, E.D. 1990. Diagnosis of Campylobacter pylori infections: the ”Gold Standard” and the alternatives. Rev Infect Dis 12 (suppl.1): S107-S114. 9. Kokkola A., Rautelin H., Puolakkainen P., Sipponen P., Farkkila M., Haapiainen R., Kosunen T.U. 2000. Diagnosis of Helicobacter pylori infection in patients with atrophic gastritis: comparison of histology, 13C-urea breath test, and serology. Scand J Gastroenterol 35(2): 138-141 10. Vaira, D., Holton, J., Menegatti., M., Ricci, C., Landi, F., Ali, A., Gatta, L., Acciardi, C., Farinelli, S., Crosatti, M., Berardi, S., Miglioli, M. 1999. New immunological assays for the diagnosis of Helicobacter pylori infection. Gut 45 (suppl. 1): I23-I27. 11. Vaira D., Miglioli M., Mule P., Holton J., Menegatti M., Vergura M., Biasco G., Conte R., Logan R.P., Barbara L. 1994. Prevalence of peptic ulcer in Helicobacter pylori positive blood donors. Gut 35: 309-312. 12. Graham D.Y., Malaty H.M., Evans D.G., Evans D.J. Jr., Klein P.D., Adam E. 1991. Epidemiology of Helicobacter pylori in an asymptomatic population in the United States. Effect of age, race, and socioeconomic status. Gastroenterology 100: 1495-1501. 13. Makristathis A., Barousch W., Pasching E., Binder C., Kuderna C., Apfalter P., Rotter M.L., Hirschl A.M. 2000. Two enzyme immunoassays and PCR for detection of Helicobacter pylori in stool specimens from pediatric patients before and after eradication therapy. J Clin Microbiol 38(10): 3710-3714

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