Helicobacter pylori Infection, Its Laboratory Diagnosis, and Antimicrobial Resistance: a Perspective of Clinical Relevance

Abstract

Despite the recent decrease in overall prevalence of Helicobacter pylori infection, morbidity and mortality rates associated with gastric cancer remain high. The antimicrobial resistance developments and treatment failure are fueling the global burden of H. pylori-associated gastric complications. Accurate diagnosis remains the opening move for treatment and eradication of infections caused by microorganisms. Although several reports have been published on diagnostic approaches for H. pylori infection, most lack the data regarding diagnosis from a clinical perspective. Therefore, we provide an intensive, comprehensive, and updated description of the currently available diagnostic methods that can help clinicians, infection diagnosis professionals, and H. pylori researchers working on infection epidemiology to broaden their understanding and to select appropriate diagnostic methods. We also emphasize appropriate diagnostic approaches based on clinical settings (either clinical diagnosis or mass screening), patient factors (either age or other predisposing factors), and clinical factors (either upper gastrointestinal bleeding or partial gastrectomy) and appropriate methods to be considered for evaluating eradication efficacy. Furthermore, to cope with the increasing trend of antimicrobial resistance, a better understanding of its emergence and current diagnostic approaches for resistance detection remain inevitable.

Keywords: Helicobacter pylori; RT-PCR; antimicrobial resistance; laboratory diagnosis; rapid urease test; stool antigen test; urea breath test.

FIG 1

H. pylori-associated pathogenicity. After ingestion, H. pylori enters the stomach, and the urease produced by the bacteria hydrolyzes the urea, thereby generating CO₂, which can be detected by the urea breath test (UBT), and ammonia. Ammonia neutralizes the acidic pH, creating an almost neutral microenvironment around bacterial cells that enables the bacteria to survive under adverse gastric conditions. Later, the bacteria find their way into the mucus layer owing to multifactorial mechanisms such as their helical shape, the presence of flagella, and chemotaxis. Several proteins (such as BabA, SabA, and OipA) produced by bacteria help in the colonization and persistence of infection. Moreover, these proteins are detected in stool specimens of infected patients by stool antigen tests (SATs). The immune response targeting numerous immunogenic proteins is evaluated by identifying antibodies using serological tests. The protein CagA is directly translocated into the gastric epithelium, and CagA-mediated carcinogenesis is triggered, whereas the VacA protein contributes to apoptosis and epithelial cell death.

FIG 2

Preferences regarding diagnostic methods. Age and clinical conditions (if any) should be considered when diagnostic methods are being selected. For children more than 10 years old, noninvasive tests, such as the urea breath test (UBT), monoclonal antibody (MAb) ELISA-based stool antigen test (SAT), serological tests, and antibody detection performed with urine specimens, are considered. For children less than 10 years old, due to immature immune response, antibody detection methods (serology and antibody detection in urine) are not considered; in such cases, the UBT or the monoclonal antibody ELISA-based SAT is the most appropriate test. The UBT and serology are considered for patients with upper gastrointestinal bleeding (UGIB), whereas for patients with partial gastrectomy, monoclonal antibody ELISA-based SAT is recommended. In patients with high risk of developing gastric cancers (*, those having active or history of PUD, low-grade gastric MALT lymphoma, history of endoscopic resection of early gastric cancer, or age over 60 years and those belonging to a family with a history of gastric cancer or a population at high risk for gastric cancer), endoscopy-based detection methods are recommended. Molecular methods, such as RT-PCR, can be conducted on specimens such as biopsy specimens, gastric juice, and stool.

FIG 3

Mass screening for H. pylori infection. Noninvasive tests, including the urea breath test (UBT), the stool antigen test (SAT), serology, or antibody detection from urine samples, are preferred methods for mass screening for Helicobacter pylori infection among communities. The positive serology results should be further confirmed by other specific methods (such as RT-PCR) on gastric biopsy or stool specimens. Patients who test positive are subjected to clarithromycin susceptibility-guided eradication therapy (when clarithromycin resistance is over 15% in that population) or clarithromycin-based empirical therapy (when clarithromycin resistance is below 15% in that population). After the completion of eradication therapy, patients are subjected to the assessment of successful eradication with either the UBT or SAT. In case of the failure of eradication therapy, a biopsy specimen-based bacterial culture is suggested to evaluate the antibiotic resistance by phenotypic (antimicrobial susceptibility testing [AST]) or genotypic (evaluation of the mutations conferring the resistance) methods, which allow selection of AST-guided therapy.

FIG 4

Mechanism of antimicrobial activity in H. pylori. (A) During bacterial multiplication, the bacterial cell wall component (i.e., the multisheet peptidoglycan layer) is synthesized by penicillin-binding proteins (PBPs), which act as transpeptidases, causing the cross-linking of the peptidoglycan polymer chains. Beta-lactams (e.g., amoxicillin) binding with PBPs via penicillin binding motifs inhibit their action, preventing the synthesis of peptidoglycan layer and leading to bacterial cell lysis and cell death. (B) The bacterial ribosomes translate the mRNA to proteins. However, the macrolides (e.g., clarithromycin) and tetracyclines bind with 50S and 30S ribosomal subunits, respectively, inhibiting protein synthesis and causing bacterial cell death. (C) In the case of nitroimidazole (e.g., metronidazole), a prodrug is activated to its active form by reductases such as RdxA, FrxA, and FrxB. The activated metronidazole damages helicoidal DNA, causing bacterial cell death. (D) DNA replication and transcription of gene initiates with the formation of a replication fork, which creates supercoiled DNA with high tension in DNA strands. DNA gyrases (A and B) cause the unwinding of supercoiled DNA which is important for the normal DNA replication and transcription by RNA polymerases. Fluoroquinolones (e.g., levofloxacin) bind with DNA gyrases (A and B), whereas the rifamycins (e.g., rifabutin) bind with RNA polymerases and inhibit their respective functions, leading to bacterial cell death.

FIG 5

Antimicrobial resistance in H. pylori. (A) Mutations leading to amino acid alterations in or around the penicillin binding motifs cause the inability of β-lactams (e.g., amoxicillin) to bind with altered PBPs. Therefore, in the presence of beta-lactams, cell wall synthesis continues, leading to bacterial resistance to beta-lactams. (B) Point mutations in the specific regions of domain V of 23S rRNA lead to the inhibition of binding of macrolides (e.g., clarithromycin) to the 50S ribosomal subunit. Therefore, even in the presence of macrolides, the synthesis of protein continues, constituting bacterial resistance to macrolides. Similarly, mutations in 16S rRNA prevent the binding of the tetracyclines to the 30S ribosomal subunit. In this way, bacterial protein synthesis continues in the presence of tetracyclines, indicating bacterial resistance to tetracyclines. (C) Due to the mutational change in the nitroreductases (RdxA, FrxA, and FrxB) the nitroreduction activity is inhibited and the nitroimidazole (e.g., metronidazole) prodrug is not activated to its functional form. Therefore, the bacterial cells become unsusceptible to the nitroimidazole antibiotics. (D) Mutations in DNA gyrases (specifically in the QRDR) prevent the binding of fluoroquinolones (e.g., levofloxacin) to the target sites. Therefore, bacterial DNA replication continues in the presence of fluoroquinolones, leading to bacterial resistance to fluoroquinolones. Similarly, mutations in rpoB leading to alterations in RNA polymerases prevent the action of rifamycins (e.g., rifabutin), resulting in bacterial resistance to rifamycins.