Large-Scale Evaluation of ureC (glmM) and SSA Conventional PCR for Rapid Direct Detection of Helicobacter pylori in Gastric Biopsies as Compared to rpoB-based Quantitative Real-Time PCR

1. INTRODUCTION

Helicobacter pylori (H. pylori) infection is one of the most widespread infections worldwide [1, 2]. It is considered a leading cause of chronic gastritis, peptic ulcer disease, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma [3-6]. Early diagnosis of this infection is crucial to establishing an effective management plan and administering appropriate antibiotic therapy to eradicate this gastric pathogen and avoid its sequelae. H. pylori diagnostic approaches are invasive methods that are based on gastric biopsies and include urease tests, histopathology, culturing, polymerase chain reaction (PCR), or non-invasive methods that utilize the urea breath test, serological detection of antibodies, and antigen detection in stool [7-10].

PCR-based methods have great utility in the detection, genotyping, and antimicrobial resistance studies of H. pylori [11-13]. These methods include many conventional and real-time varieties that can be performed directly on gastric biopsy specimens. Overall, the main advantages of these molecular-based methods are higher sensitivity, specificity, avoidance of the time required for culture-based methods, and the ability to detect dead or inhibited bacteria resulting from the prior administration of antibiotics.

Both ureC and SSA PCRs are among the earlier conventional PCR assays used for H. pylori detection [14, 15]. The ureC PCR targets the urease C (ureC) gene, which was first wrongly believed to be associated with urease production [14], but later, it was proven to be responsible for the production of the phosphoglucosamine mutase, which is involved in the synthesis of bacterial cell walls, and renamed the glmM gene [16]. SSA PCR utilizes primers targeting the 26-kDa species-specific antigen (SSA) gene [15, 17].

An earlier study was carried out to compare conventional PCR methods used for the detection of H. pylori in gastric tissues, including these two PCRs, and demonstrated higher sensitivity and specificity for ureC PCR over SSA PCR [18]. However, this study used a limited number (50 cases) of gastric biopsy specimens and took the culture method as the gold standard for comparison, which can be biased by false negative cases resulting from dead or inhibited bacteria. The current study reevaluated and compared these two PCR methods in the presence of different test conditions either tested population or test methodology, to investigate if these differences could affect the sensitivity, specificity and diagnostic accuracy of these PCR methods. The study compared ureC (glmM) PCR and SSA PCR on a wide-scale using a large number of gastric biopsies (exceeding 400 specimens) obtained from dyspeptic Saudi patients and taking rpoB real-time PCR, which targets the RNA polymerase beta-subunit (rpoB) gene, as the gold standard to avoid false negative results of culture. The principal aim was to evaluate the diagnostic value and utility of these simple PCR approaches in the direct detection of H. pylori in gastric biopsies, aiming at a rapid and accurate diagnosis of H. pylori infection, which will serve to establish appropriate treatment plans for this common pathogen.

2. MATERIALS AND METHODS

3. RESULTS

A total of 402 gastric biopsy specimens were tested for the presence of H. pylori using ureC (glmM) PCR, SSA PCR, and rpoB quantitative real-time PCR, which was considered the gold standard test.

3.2. ureC (glmM) PCR

The ureC (glmM) PCR resulted in an amplified product of approximately 294 bp in size Fig. (1). Of 402 specimens, 119 (29.6%) were H. pylori-positive, while the remaining 283 (70.4%) were H. pylori-negative (Table 1).

Table 1.

		rpoB-based quantitative real-time PCR			Sensitivity	Specificity	Positive Predictive Value	Negative Predictive Value	Diagnostic Accuracy
		+ve (n)*	-ve (n)	Total (n)
ureC (glmM) PCR	+ve	116	3	119	62% (54.9-68.68)	98.6% (95.98-99.52)	97.5% (92.85-99.14)	74.9% (69.55-79.61)	81.59% (77.51-85.08)
	-ve	71	212	283
Total		187	215	402
SSA PCR	+ve	125	1	126	66.8% (59.82-73.19)	99.5% (97.41-99.92)	99.2% (95.64-99.86)	77.5% (72.25-82.06)	84.33% (80.45-87.56)
	-ve	62	214	276
Total		187	215	402

95% CI for each diagnostic value is presented in the brackets.

4. DISCUSSION

Routine conventional PCR methods play a major role in the direct detection of H. pylori in clinical specimens, with the advantage of saving time, compared to culture-based methods. However, the different varieties of these routine PCRs differ in specificity and sensitivity, which could affect the accuracy of the diagnosis of H. pylori infection.

This study compared two types of routine PCRs, namely ureC (glmM) PCR and SSA PCR, with the gold standard rpoB gene-based quantitative real-time PCR assay. The aim of these comparisons was to investigate the diagnostic accuracies of these conventional PCRs in the direct detection of H. pylori in gastric biopsies obtained from dyspeptic patients under test conditions different from previous studies, a namely large number of tested clinical specimens, patients from different geographic region, different DNA extraction methodology, and different gold standard.

The quantitative real-time PCR assay used in our study was taken as the gold standard because it is highly specific and sensitive for the detection of H. pylori [21]. It is highly specific because it targets the RNA polymerase beta-subunit (rpoB) gene of H. pylori using a primer-probe-based approach (both the primer and probe provided with the kit are highly specific for H. pylori). As stated by the manufacturer, this kit can detect all H. pylori strains as the primers represent 100% homology with over 95% of the NCBI database reference sequences available at the time of kit design. Moreover, it is highly sensitive; by generating a standard curve using the positive control H. pylori DNA template provided with the kit, we found that the detection limit was two copies of the target gene of H. pylori per µl of the tested DNA (data not shown). Consequently, the use of such real-time PCR assay provided an advantage over the culture method by avoiding false negative results from dead or inhibited bacteria present in tested gastric biopsies.

Both tested conventional PCR methods exhibited high specificity (approximately 100%, with less than 1% difference, 98.6% and 99.5% for ureC (glmM) and SSA PCR, respectively). These findings are in complete concordance with Bickley et al. [14] and Lage et al. [19] who confirmed the specificity of ureC (glmM) PCR because the primers used did not amplify any DNA extracted from variable bacterial species other than H. pylori, including other members of the Helicobacter genus. This could be explained by the high specificity of the primers used and conservation of their target gene sequences in most H. pylori strains [14], which enables these primers to anneal specifically to their target genes.

Our results also concur with O’Toole et al. [17] who demonstrated, after sophisticated work for isolation and characterization of this specific antigen protein by determining the DNA sequence of its gene fragment, that the species-specific antigen (SSA) is unique for all H. pylori strains. These data were later used to design specific primers targeting this genomic sequence for utilization in PCR assays, which proved to be highly specific for H. pylori detection [15, 20]. Moreover, because of the universal presence of SSA in all H. pylori strains, it was used as a marker for the presence of H. pylori, acting as a positive control. Consequently, the SSA PCR assay was utilized in some studies to check the samples that were negative for H. pylori after other PCR methods used in genotyping studies [20, 22].

Despite the high specificity of both PCRs, very few false-positive results have been recorded, with three cases and one case, for ureC (glmM) and SSA PCR, respectively. This could be due to DNA cross-contamination during biopsy specimen processing, DNA extraction, or the PCR procedure itself [14].

Our results revealed 62% and 66.8% sensitivities for ureC (glmM) PCR and SSA PCR, respectively. Similarly, other studies have demonstrated variable sensitivities for both types of PCR. Lu et al. [18] found equal sensitivities for both ureC (glmM) and SSA PCR, with a detection limit equal to approximately 50 H. pylori bacterial organisms. By increasing cycle step durations to 2 min, Lage et al. [19] improved the sensitivity of ureC (glmM) PCR to a detection limit of approximately two genomes of H. pylori. Compared to our quantitative real-time PCR gold standard, the false-negative results were clearly high, with 71 and 62 cases, for ureC (glmM) PCR and SSA PCR, respectively. Such false results can be explained by occasional sequence polymorphisms that may result from point mutations in target gene sequences [23, 24] leading to the inability of primers to anneal to their targets, or the presence of PCR inhibitors, mostly from the gastric tissue specimens [25], or low or bad quality fragmented or sheared DNA.

Overall, our results, using quantitative real-time PCR as the gold standard, revealed higher specificity, sensitivity, predictive values, and diagnostic accuracy of SSA PCR over ureC (glmM) PCR. This contrasts with the findings of Lu et al. [18], who found that ureC (glmM) PCR was equally sensitive to, but more specific than, SSA PCR, taking the culture method as the gold standard. They attributed the lower specificity of the SSA PCR method to its amplification of 10 culture-negative samples. Therefore, considering the culture method as the gold standard may be the cause behind the lower specificity of SSA PCR reported in this study, as these SSA PCR-positive 10 samples may be true positive, but the culture method could not detect them. Although the culture method is considered the most specific diagnostic tool, its sensitivity to H. pylori may be low in the presence of dead or inhibited bacteria in the biopsy specimens as a result of the use of antimicrobials or improper specimen transport. The differences observed between our findings and those of others could be attributed to differences in study conditions, including the number of specimens tested, demographic and social characters of the study population, specimen preparation and processing, DNA extraction methodology, the assigned gold standard, and PCR conditions (including differences in thermocycler ramp rates).

CONCLUSION

The study reevaluated the utility of SSA PCR and ureC (glmM) PCR in the diagnosis of H. pylori infection with modification in some study conditions, including enrollment of a large number of study population from a different geographic region, different gold standard and different DNA extraction methodology. Based on our large number of tested gastric biopsies (402 non-repeated specimens) and our highly specific and sensitive gold standard (rpoB-based quantitative real-time PCR), we conclude that SSA PCR is more specific, sensitive, and diagnostically accurate than the ureC (glmM) PCR, and that the SSA PCR assay is a simple, rapid, and accurate diagnostic tool for the direct detection of H. pylori in gastric tissue specimens.

REFERENCES