INTRODUCTION
Gram-negative bacteria have been present throughout human history, and their distinctive multidrug resistance mechanisms have made them a major public health concern owing to their high morbidity and mortality rates [1]. Within this group of Gram-negative bacilli, the genus Helicobacter comprises 36 identified species, among which Helicobacter pylori is one of the most clinically significant due to its high global presence, according to studies it ranges from 24% to 73% in different populations, with a combined global prevalence of approximately 50%. H. pylori is a microaerophilic, spiral-shaped bacterium measuring 3 to 5 μm in length [2,3]. It colonizes the gastric mucosa of approximately half the global population, with the highest prevalence observed in developing countries [4]. This pathogen is associated with a wide range of gastroduodenal disorders, including chronic gastritis, peptic ulcer disease, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma [5].
The pathogenicity of H. pylori is influenced by its remarkable genetic heterogeneity; however, certain virulence-associated genes that markedly increase the risk of carcinogenesis have been identified. Cytotoxin-associated gene A (CagA) encodes the CagA protein, which has been linked to the hyperproliferation of gastric epithelial cells and development of gastric adenocarcinoma [6]. CagA overexpression can lead to severe pathological consequences in infected individuals. Despite its alarmingly high prevalence, only a fraction of the infected patients progressed to severe gastroduodenal disease [7].
Epidemiological studies have reported that H. pylori infection is widespread worldwide, with particularly high rates in impoverished and developing regions [8]. In this context, the present study aimed to evaluate the molecular detection of H. pylori in stool samples from symptomatic patients who previously tested positive for the stool antigen (SAT). This study assessed the performance and methodological utility of an enrichment-based polymerase chain reaction (PCR).
METHODS
Study population and sample collection
This methodological study was conducted at the Molecular Biology Laboratory of the Research Department of the State University of Bolívar. The study included stool samples from patients who met the selection criteria and were processed using standardized molecular diagnostic protocols. Fifty-five patients with gastroduodenal symptoms (epigastric pain, dyspepsia, abdominal distension, nausea, vomiting and/or heartburn) treated at the Alfredo Noboa Montenegro Hospital and the LabBioMedical Clinical Laboratory were included in the study. Patients who received antimicrobial or proton pump inhibitor treatment in the previous two weeks were excluded from the study.
Fecal samples were collected in sterile containers and were stored at 4°C immediately after collection to preserve the integrity of the biological material. The initial processing was performed within the first 24 h after collection. For subsequent analysis or repeat testing, aliquots were stored at -20°C until use.
SAT for initial diagnosis
The initial diagnosis of H. pylori infection was made using a Helicobacter pylori Ag Rapid Test Cassette (Healgen, GCHP-602a), following the manufacturer’s protocol. Each sample was tested in duplicate and those that yielded positive results for the H. pylori were selected.
The fecal antigen detection test was performed as an initial screening procedure in patients with a clinical suspicion of H. pylori infection due to the presence of gastroduodenal symptoms compatible with gastritis or dyspepsia. The indication for the test responded to the medical criteria and was aimed exclusively at symptomatic patients who visited for consultation. Asymptomatic volunteers were not included.
Sample enrichment and cultivation under controlled conditions
Microaerophilic cultures were performed using buffered peptone water medium (Neogen Corporation, 7148A) dissolved in type II water. The medium was distributed into flasks, and an H. pylori-positive fecal sample was added. The flasks were incubated under microaerophilic conditions (10% CO2, 5% O2, and 85% N2) using an AnaerojarTM atmospheric generation system (OXOID, iAG25) accompanied by the Anaerocult Mikrobiologie reagent (Merck KGaA 64271) for 24 hours at 37°C.
PCR was not performed directly on the fecal samples. Instead, the samples underwent a pre-enrichment culture step prior to DNA extraction to enhance bacterial recovery and DNA yield.
DNA extraction
After the microaerophilic culture phase, 2 mL of the culture was removed for DNA extraction using the Genejet Genomic DNA Purification Kit 50 RX (Thermo Fisher Scientific, K0721, USA) following the manufacturer’s protocol. DNA concentration was subsequently measured using a microspectrophotometer (Thermo ScientificTM, NanoDropTM).
PCR amplification of the 16s rRNA gene
The PCR amplification was performed in a final reaction volume of 50 μL. Each reaction contained 25 μL of Dream-Taq PCR Master Mix 2X (Thermo Fisher Scientific, K1081, Lithuania), 0.5 μM of each primer, and nuclease-free water. The target region corresponded to a 1200 bp fragment of the 16S rRNA gene, as previously described by Huang et al. [9] The primer sequences used were: Forward 5´-GTCATGACGGG TATCC-3´ and Reverse 5´-ACTTCACCCCAGTCGCTG-3´.
To improve reproducibility, the protocol was standardized, and all samples were processed under identical thermal cycling conditions. PCR was carried out using the following program: initial denaturation at 94°C for 2 min; followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 2 min; with a final extension step at 72°C for 10 min.
Quantification of genetic material
The products generated in the two amplification reactions were analyzed by horizontal electrophoresis on a 2% agarose gel (Caisson, A049-100GM) prepared in 1X TAE buffer and subjected to 100 V for 60 min. A 150 bp molecular weight marker (Promega Corporation, G210A) was used to estimate the size of the amplified DNA fragments. After running the gel, it was stained with DiamondTM nucleic acid dye (Promega Corporation, H118A) at a ratio of 1:1000 and left to act for 60 min. Finally, the fragments were visualized using an ultraviolet transilluminator.
This study was approved by the Research Ethics Committee of the State University of Bolívar, Alfredo Noboa Montenegro General Hospital (approval number: HGANM-UEB2022013). The requirement for informed consent was waived due to the retrospective nature of the study.
RESULTS
The study included 55 patients with gastroduodenal symptoms who met the eligibility criteria. Their main demographic and clinical characteristics are summarized in Table 1.
The results obtained from the antigen screening indicated that 20 of the 55 samples analyzed were positive for H. pylori infection. At the microaerophilic culture stage, only samples that tested positive for H. pylori were used. At the end of the incubation period, bacterial growth was evident in all the cultured flasks. After the extraction stage, DNA was quantified using spectrophotometry, and concentrations ranging from 3.0 to 22.1 ng/μL. Regarding the products obtained in the first amplification, only 14 of the 20 samples analyzed were positive for H. pylori and contained a 1200 bp fragment of the 16s rRNA gene. Figs. 1 and 2 show the bands obtained during the detailed visualization of the fragments in the agarose gel. The sample that showed amplification was used as a positive control. Therefore, the positivity of H. pylori infection obtained was 70%.
DISCUSSION
The pathogen H. pylori is highly prevalent worldwide, making it a significant gastrointestinal health concern. Several diagnostic methods, including SAT, are available for this purpose. This method has gained popularity in resource-limited countries as a valuable tool for identifying pathogenic infections. Although SAT is a rapid and noninvasive alternative, its effectiveness in detecting H. pylori infection is not always optimal [10].
In the present study, the SAT yielded 20 positive samples. Subsequent PCR amplification targeting the 16S rRNA gene was confirmed in 14 cases. This discrepancy in the six samples may reflect potential false positives in the antigen test or, alternatively, false negatives in the PCR assay, as both scenarios are possible, depending on the test sensitivity and sample conditions.
These findings suggest that the initial diagnostic method exhibited a precision bias related to its sensitivity, which can be explained by the immunochromatographic principle on which the test is based.
Sabbagh et al. [11], in their study on diagnostic methods for Helicobacter pylori infection, point out that the fecal antigen test (SAT) is mainly based on two types of assays: enzyme immunoassay (EIA) and immunochromatography. They reported that EIA-based tests provided more accurate and reliable results than immunochromatography-based tests. This is supported by Korkmaz et al. [12], who compared the diagnostic accuracy of different SATs for H. pylori infection and found that an EIA-based test achieved a sensitivity of 92%, whereas an immunochromatography-based test showed a sensitivity of 68.9%.
Therefore, although immunochromatography-based tests remain useful, other diagnostic techniques offer higher sensitivity and may be more appropriate for confirming H. pylori infections. Among these techniques is PCR, which has a sensitivity exceeding 95%. An additional advantage of this method is the possibility of targeting multiple genes in a multiplex PCR approach. In the present study, amplification focused on the H. pylori 16S rRNA gene.
The results of this study showed a positivity rate of 70% for H. pylori in the feces of patients with gastroduodenal symptoms using the enrichment-based PCR method. It should be noted that this figure refers to the positivity in the sample studied and does not necessarily constitute the prevalence of infection in the general population.
When compared with other studies conducted in Ecuador, we found that previous research reported substantially high infection rates. For example, a study by Bustos-Fraga et al. [13] estimated a positivity rate of 62.7% using PCR or histology.
Another report on asymptomatic patients in Ecuador indicated the presence of approximately 47.66% using antigen testing [14].
These data suggest that H. pylori infection is highly common in the country and that the 70% rate found in our study (in a subgroup with clinical symptoms) could be within the expected range for a high-risk cohort.
Factors such as limited access to drinking water, poor sanitation, degree of urbanization, and socioeconomic status can influence the burden of infection. In Ecuador, the infection is more common in rural areas or areas with less health infrastructure [15].
Likewise, it is important to recognize the methodological limitations; our value of 70% reflects positivity in a specific group (patients with gastroduodenal symptoms) and not in a representative sample of the general population. Furthermore, although the protocol used enrichment and high-throughput PCR, it does not exempt from the possibility of false negatives (for example, due to low bacterial load or inhibitors in feces) or false positives (due to contamination, cross-reactivity, or other reasons). Therefore, although these results are alarming and reinforce the need to strengthen diagnostic strategies, caution is required in their interpretations.
Given this panorama, strengthening diagnostic methods (including high-throughput non-invasive approaches, antimicrobial resistance monitoring, and early detection programs) is crucial to prevent severe complications such as peptic ulcers or gastric cancer. In particular, in high-positivity contexts such as ours, a screening strategy aimed at symptomatic patients and high-risk populations may be a viable avenue for timely intervention.
The present study had certain limitations, of which the relatively small sample size, which restricts the generalizability of the findings, is notable. As this study focused on methodological assessment rather than population prevalence, the results should be interpreted within the context of the specific cohort analyzed [16]. Another limitation was the lack of prior segmentation of patients according to risk factors and sociocultural variables. Finally, it is important to note that regardless of the detection method used, the specificity and sensitivity of the chosen test should be carefully evaluated. Despite these limitations, our results provide valuable insights into the clinical and epidemiological implications of H. pylori infection.
In conclusion, positive H. pylori PCR detection was 70%, indicating that molecular testing of fecal samples is an effective noninvasive tool for detecting infection.
High positivity rates may be associated with environmental and socioeconomic factors, highlighting the need for strengthened public health strategies for prevention and control.
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