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OXOID PYLORI TEST

Oxoid Pylori Test is a rapid latex agglutination test for the qualitative detection of Helicobacter pylori total antibodies in serum as an aid in the diagnosis of infection by Helicobacter pylori. The product is intended for use to test patients 18 years and older with symptoms of gastrointestinal disorders.
For in vitro diagnostic use.

Summary and explanation
In 1983, Marshall and Warren cultured a new pathogen from patients with gastritis¹. These findings stimulated research into the relationship between Helicobacter pylori and gastric disease. Studies have established that Helicobacter pylori (formerly Campylobacter pylori) can cause chronic gastritis², and an increasing amount of evidence indicates that there is an association between H. pylori infection and peptic ulcers^{3, 4, 5}. Recently Helicobacter pylori has also been identified as a risk factor for gastric cancer^6,7. Nearly 100% of patients with duodenal ulcers^8,9, 70% of those with gastric ulcer^2,10,11 and more than 80% of patients with gastric cancer^6,12 have Helicobacter pylori infection.
Once infection caused by Helicobacter. pylori has been diagnosed, the patient can be treated with antimicrobial drugs. Successful eradication of Helicobacter pylori leads to disappearance of gastric inflammation¹³. Among duodenal ulcer patients, eradication of Helicobacter pylori has been followed by healing of the ulcer and by reduced rate of  ulcer relapse^14,15. Several techniques, both invasive and non-invasive, are now available for diagnosing Helicobacter pylori infection. The invasive methods include culture, histological examination and urease testing of biopsy specimens. They require the collection of multiple pinch biopsy samples taken during upper gastrointestinal endoscopy. Although commonly used, the invasive methods are rather tedious and time consuming, and require a sampling procedure that may cause patient discomfort. In addition, Helicobacter pylori is a delicate organism, and is therefore easily destroyed if transported.
The non-invasive methods available include the urea breath test requiring patient ingestion of carbon isotope derivatives of urea^16,17,18 and serological detection of serum antibodies to Helicobacter pylori^19,20,21.
Helicobacter pylori elicits a specific serological response in the infected person and detection of antibodies to Helicobacter pylori in the patient's serum is a reliable indicator of Helicobacter pylori infection²². Serology has also proven to be a useful tool for monitoring efficacy of antimicrobial treatment²².

Principle of the procedure
Oxoid Pylori Test Latex Reagent containing latex particles sensitised with partially purified Helicobacter pylori antigens, is dried on the test card as dry spots. Helicobacter pylori antibodies present in serum specimens will react with the sensitised latex particles, resulting in visually detectable agglutination. Sera containing antibodies reactive to Helicobacter pylori, and sera free of antibodies to Helicobacter pylori, are included in the kit as positive and negative controls respectively.

Materials provided
Pylori Test DR0130M Kit contents (24 tests)
1. Pylori test cards 4 packs of 2 cards
2. Positive Control 1 vial
3. Negative Control 1 vial
4. Dilution Buffer 1 bottle
5. Mixing Sticks 30
6. Plastic storage pouch 1

1. Test Cards (DR0131M)
Four card packages, each containing two test cards with three reaction circles. Latex reagent sensitised with partially purified Helicobacter pylori antigens, and containing <1% sodium azide, is dried on the reaction circles.
2. Positive Control (DR0133M)
One vial (0.5 ml) of diluted rabbit serum having antibodies reactive to Helicobacter pylori, and containing < 0.1% sodium azide as a preservative. This reagent is supplied ready for use. Allow the reagent to warm to 18–25°C prior to conducting an assay.
3. Negative Control (DR0134M)
One vial (0.5 ml) of diluted newborn calf serum, non-reactive to Helicobacter pylori, and containing <0.1% sodium azide as a preservative. This reagent is supplied ready for use. Allow the reagent to warm to 18–25°C prior to conducting an assay.
4. Dilution Buffer (DR0132M)
One bottle (30 ml) of phosphate buffered saline, pH 7.2 ±0.1 containing <0.1% sodium azide as a preservative. The buffer is supplied ready for use, but should be allowed to warm to 18–25°C prior to use.
5. Mixing Sticks
Thirty (30) double-ended mixing sticks for mixing latex and diluted serum.
6. Plastic storage pouch
One plastic pouch for storage of the opened test card package containing unused test cards.

Materials required but not provided
Micropipette
Micropipettes capable of delivering volumes of 40 µl, 50 µl and 150 µl (see Procedure, Dilution Method A), or 10 µl and 30 µl (see Procedure, Dilution Method B) are needed.

Test Tubes
Plastic or glass tubes for diluting patient serum samples prior to testing, if using Dilution Method A.

Warnings and precautions
Health and Safety Information
1. Oxoid Pylori Test is designed for in vitro diagnostic use only and should be used by properly trained individuals.
2. Specimens may contain infectious agents. All specimens should be handled and discarded as a potential biohazard.
3. Non-disposable apparatus should be sterilised by any appropriate procedure after use, although the preferred method is to autoclave for 15 minutes at 121°C; disposables should be autoclaved or incinerated. Spillage of potentially infectious materials should be removed immediately with absorbent paper tissue and the contaminated areas swabbed with a standard bacterial disinfectant or 70% alcohol. Materials used to clean spills, including gloves, should be disposed of as biohazardous waste.
4. Do not pipette by mouth. Wear disposable gloves while handling specimens and performing the assay. Wash hands thoroughly when finished.
5. The dried latex reagent on the cards contain as preservative <1% sodium azide which is harmful when inhaled, swallowed, or in contact with skin. The dissolved latex reagent contains <0.1% sodium azide which is not considered to be a harmful concentration. The other reagents in the kit contain <0.1% sodium azide, as preservative. The copper and lead used in some plumbing systems can react with azides to form explosive salts. The quantities of azide used in this kit are small; nevertheless when disposing of azide-containing materials, they should be flushed away with a large volume of water.
6. Although the reagents do not contain Helicobacter pylori infectious agents, they are prepared from biological materials and should be handled and discarded as a potential biohazard.
7. Disposal of all specimen and test materials should be in accordance with local regulations.
8. When used in accordance with the principles of Good Laboratory Practice, good standards of occupational hygiene and the instructions stated in this

Analytical Precautions
1. Do not use the reagents beyond the indicated expiration date. Microbiological contamination of reagents must be avoided as this may reduce the life of the product and cause erroneous results.
2. Allow all reagents to reach room temperature (18–25°C) before use. Return reagents to 2–8°C for storage as appropriate, immediately after use.
3. Do not mix reagents from different lots of Oxoid Pylori Test.
4. Do not touch the reaction areas on the cards.
5. Do not freeze the kit.
6. A sticky dry spot is evidence that the reagent has been exposed to moisture and excessive heat and should not be used.

Specimen collection and storage
A normal venous blood sample should be taken and the serum separated. If serum is not immediately analysed, it can be stored at 2–8°C for seven (7) days or at –20°C or lower temperature for several months. (Do not store in self-defrosting freezers.) Before opening, all components should be stored at 2–8°C where they will retain their activity until the date shown on the label. Opened test card packages containing unused test cards must be stored in the plastic storage bag provided, taking care to seal the bag tightly (do not remove the silica gel pouch from the card package!). The card package can be used for five (5) weeks after it has been opened if stored properly.

Procedure
Remove the reagents from the refrigerator and allow them to reach room temperature (18–25°C) before use.
For proper performance of the test, dilute patient samples with Dilution Buffer prior to testing (Positive and Negative Control reagents are used directly in the test without dilution).
The dilution step can take place either separately in a glass or plastic test tube (Dilution Method A) or directly on the test card (Dilution Method B). Both Dilution Methods give equivalent test results.

Dilution Method A (Dilution in a Test Tube)
1. Dilute the serum specimen 1:4 with Dilution Buffer in a test tube (e.g. 50 µl specimen + 150 µl buffer).
2. Pipette 40 µl of diluted serum sample onto a circle of the test card next to the latex reagent. Proceed as described in “Use of Dry Latex Test Cards”.

Dilution Method B (Dilution on the Test Card)
1. Using a micropipette, dispense 30 µl of Dilution Buffer next to the latex reagent onto one circle of the test card for each sample to be tested.
2. Dispense 10 µl of patient serum sample to be tested onto the Dilution Buffer drop. Mix thoroughly by pulling the solution back and forth into the pipette at least five (5) times. Proceed as described in “Use of Dry Latex Test Cards”.

Use of Dry Latex Test Cards
1. When testing either the Negative or the Positive kit Control, dispense one drop from the respective dropper vial onto a clean circle of the test card.
Note: the Control Reagents are already diluted and are used as dispensed.
2. Using the clean end of a mixing stick for each circle, carefully mix the samples or controls with the latex reagent on the card. Use the sample to cover the entire circle. Discard each stick after use.
3. Tilt and rotate the test card, moving the reagents in a circular motion within the circle. Observe the latex particles for evidence of agglutination occurring within
three (3) minutes.

Reading of results and interpretation
The test result is reactive, i.e. the serum sample contains detectable level of antibodies to Helicobacter pylori, when agglutination is detected in the test circle within three (3) minutes. The agglutination may be complete (red granules on a white background) or partial (granules can be detected but the background remains opaque). The test result is nonreactive and the sample does not contain detectable level of antibodies to Helicobacter pylori when no agglutination is detected in the circle within three (3) minutes. A positive test result does not allow one to distinguish between active infection and colonisation of Helicobacter pylori.
If controls do not behave as expected, assay results are invalid.

Quality control
To check the performance of the kit reagents use the provided Positive and Negative Control instead of the patient sample. The controls should be performed each day the Pylori test is used. Follow the procedure as described in “ Procedure”.
To check the performance of the Dilution Buffer follow the procedure as described in the “ Procedure” section replacing the diluted sample with 40 µl of Dilution Buffer.
The inability of the Positive Control to yield agglutination within three minutes, or any evidence of a non-specific agglutination with either the Negative Control or the Dilution Buffer is regarded as evidence of reagent deterioration. Such reagents must not be used for patient samples. The controls should not be used if they are contaminated.

Limitations of the procedure
1. The test results should be weighed along with the patient’s clinical symptomology in determining its significance.
2. A negative test result indicates that the patient does not have detectable levels of antibodies to Helicobacter pylori. This may occur when testing a patient at too early a stage in the development of disease, before an immune response is mounted.
3. A positive test result does not distinguish between the presence of active or passive disease or colonisation in the patient.
4. A positive test result does not necessarily indicate a gastrointestinal disorder.
5. The test should be carried out only for patients symptomatic for gastrointestinal disorders and is not intended for use with asymptomatic patients.
6. The performance of this assay has not been established for patients under 18 years of age.
7. Performance has not been demonstrated for a correlation of detection of antibody and the presence of Helicobacter pylori, therefore no association can be made to the presence of Helicobacter pylori or infectivity.

Expected values
There are only a few studies concerning Helicobacter pylori infections in the asymptomatic populations due to the fact that the diagnostic methods available (endoscopy and biopsy) are invasive procedures. Siurala²³ and Kekki²⁴ showed in their studies the occurrence of gastritis in the asymptomatic population in Finland and Estonia, that the frequency of gastritis increases with age. Among the 30-year-old population, the incidence was 30% and increased to over 70% by the age of 60. Later, Petross²⁵, Rauws²⁶ and Barthel²⁷ observed that 20% of young asymptomatic adults (mean age 30 years) suffered from histologic gastritis, associated with Helicobacter pylori infection. The frequency of histologically confirmed gastritis in a normal population correlates well with the occurrence of Helicobacter pylori specific antibodies. This has been confirmed in studies of Helicobacter pylori antibody levels in asymptomatic populations by non-invasive serological methods^28,29.

Table 1 shows the prevalence of Helicobacter pylori antibodies found with Oxoid Pylori Latex Test in 500 asymptomatic Finnish blood donors, aged 18–65 years30.

Percentage of Finnish Blood Donors in different age groups with Helicobacter pylori antibodies.

The incidence of Helicobacter pylori antibodies found with Oxoid Pylori Latex Test in patients at different diagnostic status is presented in Table 2. Serum samples of 195 patients with upper abdominal complaints were tested³⁰.

The incidence of Helicobacter pylori antibodies found with Oxoid Pylori at different diagnostic status.

N* = the number of each different diagnostic status, only one type or multiple types of diagnostic conditions could be found in the patients. Most often at least two or more diagnostic conditions were diagnosed.

Specific performance characteristics
Study I
Comparison of the performance of Oxoid Pylori Test against the results obtained with the reference method including culture, acridine staining and giemsa staining.
The Oxoid Pylori Test was evaluated using serum samples from 195 patients with upper gastrointestinal symptoms attending a gastroenterology clinic³⁰. The results are presented in Table 3.
The results were compared with results obtained with a reference method including culture, acridine staining and giemsa staining. The reference method was considered positive if at least one of the tests included was
positive for Helicobacter pylori. The reference method was considered negative if all of the tests included were negative.

Correlation between Oxoid Pylori Test and the reference tests.

Sensitivity 97% (91.9% to 99.4%)*
Specificity 85% (76.3% to 92.0%)*
Agreement 92%
*Represents a 95% confidence limit.

Analysis of discrepant results
Discrepant results were confirmed by immunoblot procedure. Of 13 reference method negative/Oxoid Pylori Test positive samples, eleven contained specific antibodies to Helicobacter pylori when tested by immunoblot. The three reference method positive/Oxoid Pylori Test negative samples were all negative by immunoblot, these samples would still be considered reference method positive samples.

Table 4 Correlation between biopsy data and Oxoid Pylori Test.

*Culture and/or acridine-orange stain and/or giemsa stain positive.
The correlation between biopsy data (chronic gastritis +/- and H. pylori +/-) and Oxoid Pylori shown in Table 4. The status of chronic gastritis is interpreted on the findings of the pathologist and the presence of Helicobacter pylori based on positive results either from culture, or acridine staining or giemsa staining³⁰.

Study II
Comparison of performance of Oxoid Pylori Test against the results of culture and an enzyme immuno-assay (IgG).
A comparison study between the Oxoid Pylori Test, culture and an in-house EIA procedure was performed³⁰. Serum specimens were collected from 691 adult patients with gastrointestinal symptoms. Biopsies from 688 patients were cultured for Helicobacter pylori.
The EIA procedure used is a microtitre plate based assay using a glycine extracted antigen. Antibodies present in patient serum samples were detected with alkaline phosphatase conjugated anti-human lgG conjugate.
Helicobacter pylori bacteria was detected in 264 out of 688 biopsies cultured. Of these patients 97.7% were found positive with Oxoid Pylori Test. The results are presented in Table 5.

Comparison between Oxoid Pylori Test and culture.

Sensitivity 98% (95.1% to 99.2%)*
Specificity 81% (77.4% to 84.9%)*
Agreement 88%
*Represents a 95% confidence limit.

Of 6 culture positive/Oxoid Pylori Test negative samples, three were also EIA positive (2 lgG and 1 lgA positive). Among the 80 culture negative/Oxoid Pylori Test positive specimens, 50 were EIA positive too (20 IgG and 5 IgA only, and 25 both lgG and IgA positive).
The results of the comparison of Oxoid Pylori Test with EIA (IgG) procedure is presented in Table 6. Oxoid Pylori Test detected 97.3% (291/299) of the EIA positive sera. Two out of eight EIA (lgG) positive/Oxoid Pylori Test negative samples were positive in culture. In addition, 14 out of the 49 EIA (IgG) negative/Oxoid Pylori Test positive sera were positive in culture, and five out of the 49 were lgA positive.

Note for tables 6, 7 & 8. Please be advised that “relative” refers to the comparison of the Oxoid test to that of a similar assay. There was no attempt to correlate the assay’s results with presence/absence of disease. No judgement can be made on the comparison assay's accuracy to predict disease.

Table 6
Comparison between Oxoid Pylori Test and EIA (IgG).

Relative Sensitivity 97% (94.8% to 98.8%)*
Relative Specificity 88% (84.2% to 90.8%)*
Agreement 92%
*Represents a 95% confidence limit.

Study III
Comparison of the performance of Oxoid Pylori Test against the results of a commercial Pylori ELISA test.

III/a. 182 sera from the Study I were examined with Oxoid Pylori Test and a commercial Pylori ELISA test kit. The results are presented in Table 7.
 
Comparison between Oxoid Pylori Test and a commercial Pylori ELISA test.

Relative Sensitivity 88.0% (82.2% to 93.9%)*
Relative Specificity 96.8% (89.0% to 99.6%)*
Agreement 91.1%
*Represents a 95% confidence limit.
Test results of two (2) sera were excluded from the calculation of sensitivity, specificity and agreement because of the equivocal results obtained by the commercial Pylori ELISA Test kit.

III/b. 274 out of 691 patient serum samples from the Study 11 were evaluated with a commercial Pylori ELISA test. The results were compared with those obtained with Oxoid Pylori Test. The results are presented in Table 8.
 
Comparison between Oxoid Pylori Test and a commercial Pylori ELISA test.

Relative Sensitivity 96.5% (92.2% to 98.9%)*
Relative Specificity 95.3% (90.0% to 98.2%)*
Agreement 95.9%
*Represents a 95% confidence limit.
Four (4) test results were excluded from the calculation of sensitivity, specificity and agreement because of the equivocal results obtained by commercial Pylori ELISA test kit.

Bacterial cross reactivity of Oxoid Pylori Test
The specificity of the Oxoid Pylori Test was tested by absorption of patient samples with antigen extracts from various bacterial species. Aliquots of each of two patient sera, confirmed positive by biopsy culture, were pretreated with sonicated antigen extracts (0.3 mg/ml) from a panel of bacterial species. After incubation the samples were retested with Oxoid Pylori Test. The results are presented in Table 9.
 
The specificity of Oxoid Pylori Test.

The results show that the reactivity of Oxoid Pylori Test was inhibited in both samples after absorption of the sera with Helicobacter pylori antigen. Absorption with antigen extracts from the other tested bacteria had no effect on the results of Oxoid Pylori Test. This indicates that Oxoid Pylori Test does not cross-react with any of the tested bacteria.

Reproducibility of Oxoid Pylori Test
Three serum samples were analysed 10 times on three days using a fresh dilution of each serum each day. One of the three sera was negative (titre 110), one was borderline positive (titre 690) and one was high positive (titre 9380) when tested with Pyloriset EIA-G. The cutoff value of the EIA test is 500. Oxoid Pylori Test kits from the same lot number were used for all testing. The results are presented in Table 10.

Reproducibility of Oxoid Pylori Test.

The good agreement of results in repeated testing on each day suggests that the test performance as well as the interpretation of results is highly reproducible.

References.
1. Warren JD, and Marshall BJ (1983). Lancet, i:1273–1275.
2. Marshall BJ and Warren JR (1984). Lancet, i:131 I–1314
3. Johnston BJ et al. (1986). Gut, 27: 1132–1137.
4. Goodwin CS (1988). Lancet. ii: 1467–1469.
5. Rathbone BJ et al. (1986). Gut, 27: 635–641.
6. Parsonnet J et al.(1991). N. EngI. J. Med., 325:1127–1131.
7. Forman D et al. (1990). Int. J. Cancer, 46:608–611.
8. Graham DY (1989). Scand. J. Gastroenterol., 24 (suppl. 160): 46–52.
9. Vaira D et al.(1988). J. Clin. Pathol., 41: 355– 356
10. Price AB (1988). Scand. J. Gastroenterol., 23 (suppl. 142): 21–24
11. Dooly CP and Cohen H (1988). Ann. Intern. Med., 108: 70–79.
12. Nomura A et al. (1991). N. Engl. J. Med., 325: 1132–1136
13. Valle J et al. (1991). Scand. J. Gastroenterol., 26:1057–1066
14. Marshall BJ et al. (1988). Lancet, ii: 1437–1441.
15. Seppälä KM et al. (1992). Scand. J.Gastro-enterol., 27:973–976.
16. Graham DY, Klein PD, Evans DL (1987). Lancet, i: 1174–1177.
17. Marshall BJ, Surveyor l (1988). J. Nucl. Med., 219:11–16.
18. Rauws EAJ, Van Royen EA, Langenberg W (1989). Gut, 30(6): 798–803.
19. Evans DJ; Evans DG, Graham DY, Klein PD (1989). Gastroenterol., 96: 1004–1008.
20. Newell DG, Rathbone BJ (1989). Serodiag. and Immunother. Infect. Dis., 3: 1–6.
21. Evans DJ, Evans DG, Smith KE, Graham DY (1989). Infect. Immun., 57(3): 664–667.
22. Kosunen TU et al (1992). Lancet, 339: 893–895.
23. Siurala M, Isokoski M, Varis K, Kekki M (1968). Scand. J. Gastroenterol., 3:211–223.
24. Kekki M, Villako M, Tamre A, Siurala M (1977). Scand. J. Gastroenterol., 12: 321–324.
25. Pettross CW, et al. (1988). Dig. Dis. Sci., 33: 649–653.
26. Rauws EA, Langenberg W, Houthoff HJ, Zanen HC, Tytgat GN (1988). Gastroenterol., 94:3340.
27. Barthel JS, Westblom TU, Jauy AD, Gonzalez F, Everett ED(1988). Arch. Intern. Med., 148:1149–1151.
28. Jones DM, Eldridge J, Fox AJ, Sethi P, Whenwell PJ (1986). J. Med. Microbiol., 22: 57–62.
29. Dooley, CP, et. al.(1989). NewEng. J. Med.,321: 1562–1566.
30. Data on file, Oxoid Ltd