Two new FDA-cleared immunoassays added to test ...

Introduction

The ACG Guidelines recommend non-invasive tests which detect current disease to diagnosis H. pylori infection in patients under the age of 55 who are not exhibiting alarm symptoms (1). Non-invasive H. pylori testing options include stool antigen testing, urea breath testing, and serology (1, 2). Laboratory directors must decide how to optimally combine test accuracy, cost, ease-of-use and time-to-result. Two new FDA-cleared immunoassays, the first in 15 years, are now available. The strong performance of these new bench-top rapid stool antigen tests and their sample flexibility offer several advantages over alternative testing methods.

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H. pylori microbiology influences test design and choice

H. pylori bacteria are specialized for growth in the harsh acidic environment of the stomach (3, 4). The bacteria use flagella to propel the organism into and under the mucus layer that normally protects the epithelial cells of the stomach. Although this mucus layer is much less acidic that the lumen, H. pylori further adjusts the pH of its mucus niche. H. pylori expresses a urease enzyme that breaks down urea into ammonia to increase local pH while releasing carbon dioxide. This reaction is uncommon in other stomach bacteria and is the basis of some testing methods (4).

Unlike many other gastrointestinal bacteria, H. pylori typically is not cultured from fecal specimens or even from endoscopic biopsy samples unless antibiotic resistance testing is required. However, antibiotic resistance is an increasing problem (5, 6). Eradication of H. pylori can no longer be assumed after standard antibiotic therapy. This has led to adoption of a Test- Treat-Test strategy in which patients undergo follow-up testing four weeks following eradication therapy to determine if the bacteria have been successfully eliminated (1, 5, 7). A non-invasive stool antigen test or urea breath test (UBT) is favored for this follow-up testing to avoid the expense and discomfort of endoscopy and biopsy for the patient. Serology that detects antibodies to H. pylori cannot be used for follow-up, as antibodies persist even after successful eradication (1).

Patients can also influence test results

H. pylori infections and associated symptoms and inflammation can wax and wane. Patients who seek medical care for symptoms are often experiencing active inflammation and may already be taking a proton pump inhibitor (PPI) or bismuth-containing antacid. These compounds can alter the reliability of both UBTs and stool antigen tests by decreasing inflammation (1).

For this reason, UBTs require discontinuation of supportive therapies. With stool antigen testing, a positive result is reliable, a negative result in a patient still suspected of having an H. pylori infection may warrant discontinuation of supportive therapy and retesting. Not surprisingly, patients dislike stopping these supportive therapies for 10-14 days prior to testing. Compliance rates are unknown but circumstantial evidence suggests that testing laboratories may wish to request confirmation of patient PPI status.

Also, an unknown fraction of patients may have current or recent antibiotics exposure. Antibiotic treatment within four weeks prior to testing may suppress bacteria below detectable levels but not eradicate them fully. Thus, antibiotics therapy should be completed prior to these tests to avoid false negative results (1).

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According to the predefined criteria, a total of 46 (50.5%) patients tested were positive by at least 2 of the 3 biopsy-based methods. The best sensitivity (95.6%) belonged to histology and RUT. The sensitivities of other tests including PCR, serology and stool antigen test were 93.5%, 91.3% and 73.9%, respectively. RUT showed the best specificity (100%), and the specificities of the other tests, including PCR, stool antigen test, histology and serology, were 95.6%, 86.7%, 77.8% and 55.6%, respectively.

patients were included in the study, and specimens including biopsies, blood and stool were taken. Biopsies were evaluated by hematoxylin and eosin, and Giemsa staining. A sequence of 294 bp in the ureC (glmM) gene was amplified. The rapid urease test (RUT) was performed using a non-commercial validated test. Stool samples were analyzed using a polyclonal ELISA stool antigen test. A serological assay for IgG antibodies was performed by a commercial Helicobacter pylori IgG ELISA kit.

Invasive and non-invasive techniques are used to diagnose H. pylori infection. Some factors influence the choice of a diagnostic test, such as the sensitivity and specificity of the tests, the clinical circumstances and the cost-effectiveness of the testing strategy. The aim of this study was to reveal the relationship between different H. pylori infection diagnosis methods, and clarify the application scope of each diagnosis method.

The aim of the study was to reveal the relationship between different H. pylori infection diagnosis methods (Histology, RUT, PCR, stool antigen test and serology) and to clarify the application scope of each diagnosis method and its influencing factors.

UBT has greater sensitivity and specificity than other non-invasive tests but its specificity is decreased when other urease producing bacteria are present in the human gut ( 11 ). It also needs more expensive and complicated equipments.

In individuals not undergoing gastroscopy, serology is the easiest way to detect H. pylori infection by detecting circulating antibodies against H. pylori. However, it cannot differentiate between active and asymptomatic colonization and past and current H. pylori infection ( 10 ).

Bacterial culturing from gastric biopsies is regarded as a definite proof of H. pylori infection. Since the method is more technically demanding, the ability to culture and the sensitivity of the test may vary between laboratories ( 7 ). In clinical practice, the most routinely used technique is RUT. However, to obtain a sufficient sensitivity, there should be sufficient bacterial load consisting of at least 10 5 bacteria. Therefore, the test is less advisable for post-eradication follow-up since this amount may not be present after about 4 weeks of the failure of eradication therapy ( 8 - 9 ).

Invasive and non-invasive techniques are used to diagnose H. pylori infection. Invasive methods such as histology, rapid urease test (RUT), microbiological culture and polymerase chain reaction (PCR), require endoscopy and are also known as biopsy-based tests. Non-invasive tests include stool antigen test, serology and urea breath test (UBT). Some factors which influence the choice of a given testing strategy include sensitivity, specificity, the clinical circumstances and the cost-effectiveness of the test ( 5 ). Notably, all these techniques have their own limitations ( 6 ). In countries where endoscopy is frequently performed, one of the most commonly used techniques is histopathological diagnosis. Experienced pathologist and quality of biopsies are two basic requirements for the proper histopathological examination. Improper biopsies, observer related factors, topographical changes in the stomach, H. pylori density and its patchy distribution and type of stain used may cause false results ( 3 ).

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Helicobacter pylori is a microaerophilic spiral shaped gram-negative bacterium which colonizes the human gastric mucosa ( 1 ). It is regarded as the major cause of duodenal ulcers, gastric and gastritis, mucosa-associated lymphoid tissue (MALT) lymphoma and gastric adenocarcinoma. The prevalence of H. pylori infection is 25%-50% in developed countries and 70%-90% in developing countries ( 2 - 3 ). The most probable mode of transmission is person-to-person spread but oral-oral and fecal-oral transmissions have also been reported ( 4 ).

On the endoscopy day, 5 ml blood was taken from patients and transferred to the laboratory. The sera were separated and kept until the day of testing at -20°C. A serological assay for IgG antibodies against H. pylori was performed by a commercial Helicobacter pylori IgG ELISA kit (IBL, Hamburg, Germany) according to the manufacturer&#;s instructions. The results were classed as positive if anti&#;H pylori immunoglobulin (Ig) G titers were >12 U/ml, negative if they were < 8 U/ml, and equivocal if they were between 8 and 12 U/ml.

Stool samples were analyzed using a polyclonal ELISA stool antigen test (Astra s.r.l, Milan, Italy), according to manufacturer&#;s instructions. Briefly, diluted fecal samples and peroxidase-conjugated polyclonal antibodies were added to the wells. After 90 minutes of incubation at room temperature, sample wells were washed to remove unbound samples and enzyme-labeled antibodies. The results were read at 450/620nm by spectrophotometry. To determine H. pylori antigen concentrations in test samples, a cutoff value of OD 0.2 was used. Samples with OD values < 0.150 were considered negative. Samples with OD values within 0.150- were considered borderline and samples with OD values > 0.250 were considered positive ( 14 ).

DNA was extracted from biopsies using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). A sequence of 294 bp in the ureC (glmM) gene was amplified as described previously ( 13 ).Primer pair used for ureC amplification had the nucleotide sequence as follows: forward primer,(5&#;-AAGCTTTTAGGGGTGTTAGGGGTTT-3&#;) and reverse primer (5&#;- AAGCTTACTTTCTAACACTAACGC-3&#;). The PCR conditions consisted of 1 cycle of 5 min at 93°C, followed by 35 cycles of 1 min at 93°C, 30 s at 55°C, 30 s at 72°C, and a final cycle of 10 min at 72°C. Amplified products were visualized on 2% agarose gel under UV light. All assays were performed at least twice.

Biopsies from the antrum and the corpus were obtained for histology and were fixed in 10% formalin and sent to the laboratory. Paraffin embedded and multiple 4 mm-thick histological sections were obtained from each biopsy. Preparations were stained with hematoxylin and eosin, and Giemsa evaluated by several pathologists blinded to the results of the other tests. The presence of H. pylori was determined but not graded.

The RUT was performed using a non-commercial validated test. This test was performed with a homemade solution with 1 ml distilled water, one drop of 1% phenol red, and 100 mg urea, prepared just before endoscopy. One antral sample was placed in the solution and maintained at room temperature. The test was considered positive when the color changed from yellow to red within 24 hours ( 12 ).

Several biopsy specimens were obtained: one was reserved for RUT, and some, derived from the antrum and corpus, for histology (formalin-fixed and paraffin-embedded) and finally one for PCR. Stool specimens and serum samples from these patients were collected and kept on -20°C until used.

Totally, 91 patients who presented for routine upper gastrointestinal endoscopy at Firoozgar Hospital (a University hospital) and were willing to cooperate in this study were included. The patients included 39 males and 52 females with a mean age of 45 years (range, 17&#;87 years). Subjects who had received antimicrobial therapy, H2-receptor blockers, proton-pump inhibitors and non-steroidal anti-inflammatory drugs 30 days prior to endoscopy were excluded from the study. The ethics committee of the university granted approval for the study and all the patients gave their consent to participate.

The positive and negative histology results were received within the next few days. RUT results were observed within a few minutes up to 24 hours. Positive PCR results were observed on agarose gel as a band at with 294 bp in size ( ). As the culture of H. pylori from biopsies was not performed in our study, other endoscopic-based techniques (RUT, PCR and histological staining of the biopsies) were considered the gold standard for determination of the specificity and sensitivity of each test. Patients were considered to be infected with H. pylori if 2 of 3 tests were positive. Based on the stated criteria, 46 (50.5%) of the patients were diagnosed as H. pylori infected and 45 (49.5%) as uninfected.

DISCUSSION

In the clinical setting, a rapid and cost-effective detection method for diagnosis of H. pylori infection is desirable. H. pylori infection can be detected by a variety of methods (15). In the routine clinical diagnostics the urease test, histological examination, urea breath test, serology, bacterial culture and stool antigen test are valuable methods of detecting H. pylori infection.

Histopathology has historically been considered as being the first diagnostic method for H. pylori detection and is still widely used as the main diagnostic tool in suspicious patients with upper gastrointestinal symptoms or in highly prevalent areas (16). Correct and trustworthy histologically diagnosing of H. pylori gastritis has a high influence on clinical practice as a therapeutic indicator. Nevertheless, several previous studies display important inter-observer variation, suggesting that the skills of the pathologist are impressive when it comes to the histopathological diagnosis of H. pylori (17-18). In the present study, one pathologist would normally found more positive results when the other tests were negative, suggesting that the experience and skills of pathologist do matter for the specificity and sensitivity of histopathological examination (19).

In the present study, the sensitivity of rapid urease test was 95.6%, which is very close to those by other authors (14, 20-22). Also, the specificity of RUT is rather the same as those reported by other workers, though we did not get any false positive result by RUT in contrast with the other studies (14, 20, 22).

Molecular methods have the advantage of their rapidity and the limited influence of the transport conditions. To date, many PCR methods have been developed to detect directly the organism in clinical samples. A variety of genes including the cagA gene, the ureC (glmM) gene, the ureA gene, the 16S rRNA gene and the 26-kDa species-specific antigen (SSA) gene have been used as targets. Lage and colleagues showed that the ureC amplifications were obtained only with H. pylori, while none of the other urease-positive or related bacteria that had been tested gave the expected amplified DNA products (23). Our study re-confirms the sensitivity and the specificity of the PCR assay with the ureC (glmM) primers (6, 13, 23).

Many serological tests are available commercially. They are widely in use because they are inexpensive and easy to use. However, since antibody titers can remain high for months after elimination of infection (24), the sort of the tests based on the detection of specific antibodies are not reliable to check eradication of H. pylori (25). In this study, serology showed the lowest specificity and accuracy in comparison with other tests. Generally, low accuracy of IgG serological tests is due to the inability to differentiate between current and past infection. In contrast, considering that almost all previously treated participants were excluded by the questionnaire, and those H. Pylori infection was rarely cured spontaneously, 11 of the single positive serology test might reflect past infection and/or false positive test result (14).

To date, several stool antigen tests have been developed commercially. Despite the heterogeneity in reported sensitivity and specificity rates, most of them have acceptable results and many studies have claimed that the stool antigen test is useful for the primary diagnosis and post-treatment follow-up of H. pylori infection (26). Premier Platinum HpSA as the first and the most used valid H. pylori stool antigen test, has been recommended as a reliable alternative to UBT in the initial diagnosis and follow-up period (27-28); but there are a number of studies reporting a lower level of accuracy (29-30). In our study, stool antigen test showed the lowest sensitivity (73.9%), and not so good specificity (86.7%). Upon our search, we could only found one study which had used this sort of kit. Despite nearly identical conditions, their results were better than that found in our study (85% for sensitivity and 90% for specificity)(31). The accuracy of the test might change from lot to lot and intertest variability has already been reported by Makristhatiset al.(32). Therefore, such discrepancies could be assigned not only to methodological failures but also to intertest variability(33).

Based on the results provided by this study, the accuracy of the tests for H. pylori diagnosis can be arranged in order as follows: RUT >PCR>histology> stool antigen test> serology. However, the arrangement might change slightly among similar studies. But the general point is that in almost all studies, biopsy-based methods are preferred over other methods; and none of these methods can be considered as the gold standard alone. Thus, simultaneous utilization of biopsy-based and non-invasive methods is recommended for H. pylori infection confirmation.

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