Antimicrobial susceptibility testing is needed to adapt Helicobacter pylori treatment to obtain the best results. Beside the standard phenotypic methods, molecular methods are increasingly used. The value of these molecular tests is that they are quick, independent of the transport conditions, easy to standardize, and commercial kits are available. In this article, these methods are reviewed, focusing on the determination of H pylori resistance to macrolides and fluoroquinolones, and mentioning also the methods used for tetracycline and rifampin.
Key points
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Detection of antimicrobial resistance of Helicobacter pylori is important to tailor the treatment and obtain the best outcome of eradication.
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Molecular methods that detect mutations in genes relevant to antimicrobial resistance can be applied, especially for the most important antibiotic (ie, clarithromycin).
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Numerous molecular methods have been proposed to detect the main 3 mutations associated with clarithromycin resistance of H pylori , the most commonly used being real-time polymerase chain reaction protocols.
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The correlation between molecular detection of resistance via mutations and antimicrobial susceptibility testing by Etest is not perfect, because the former is better for detecting heteroresistance, but which method correlates the best with eradication is not known.
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Molecular methods can also be applied to detect H pylori resistance to fluoroquinolones, tetracycline, and rifampin, although they are not so commonly used.
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The advantage of molecular methods is their rapidity, lack of stringent transport conditions, and standardization.
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Their limit is that they cannot be used for all antibiotics and they do not detect resistance caused by mutations other than those already known or other resistance mechanisms.
There are several reasons for failure of the treatments aiming to eradicate Helicobacter pylori . They include a poor compliance to the regimen and a high gastric acidity, which is not overcome by the recommended dose of proton pump inhibitor (PPI) that increases the minimal inhibitory concentration (MIC) of the antibiotics used. In the past, different conditions, such as an important bacterial load, infection by CagA (cytotoxin-associated gene A)-positive versus CagA-negative H pylori strains, and the presence of intracellular bacteria and some immunologic deficiencies have been suggested to influence eradication but seem less important when susceptibility and compliance are taken into consideration.
H pylori may become resistant to all the antibiotics used for eradication in the various regimens proposed, essentially according to the same mechanism (ie, acquisition of point mutations). Point mutations occur by chance, and increase the MIC of the bacteria. Those organisms with point mutations are then selected by the corresponding antibiotics when prescribed. Another mechanism that sometimes occurs is an efflux mechanism of resistance (ie, efflux pumps, which tend to eliminate the antibiotic having penetrated into the bacterial cell).
Acquisition of resistance in H pylori is important essentially for macrolides (clarithromycin) and fluoroquinolones (levofloxacin). It rarely occurs for β-lactams (amoxicillin), tetracyclines, and for rifampin (rifabutin). To the contrary, although they seem to be frequent for 5-nitroimidazoles (metronidazole), they can be overcome in vivo.
As for any infection, it seems crucial to detect H pylori resistance before prescribing a treatment, the efficacy of which would be jeopardized by the presence of resistant organisms.
The standard detection method consists of performing an antibiogram, usually or MIC determination, using Etest. Although this procedure has the advantage of offering testing of all of the antibiotics of interest, it also has some drawbacks. It requires living organisms, and culturing H pylori is sometimes challenging because of the special transport conditions necessary for gastric biopsies, as well as special care in the laboratory; several days are necessary for primary culture and then performing the antibiogram. For these reasons, alternative methods to this phenotypic approach have been proposed, including various molecular approaches.
The aim of this article is to review these methods, focusing on the determination of H pylori resistance to macrolides and fluoroquinolones, which are the most important, and mentioning also the methods used for tetracycline and rifampin.
Molecular determination of Helicobacter pylori resistance to macrolides
Mechanisms
Macrolides target the 23S ribosomal RNA (rRNA). There are in particular 2 nucleotide positions at the domain V level of the peptidyl transferase loop, which can lead to resistant organisms, because they induce a change in the ribosome conformation and decrease macrolide binding. These positions are 2142 and 2143. A transition can be found at both positions, whereas a transversion is found only at the former ( Fig. 1 ). Other mutations that could theoretically occur are not found in nature, possibly because they lead to nonviable organisms. Some reports of other mutations associated with clarithromycin resistance have been made but could not be confirmed.
However, a recent study questions this dogma. Comparing phenotypic and genotypic resistance to clarithromycin, De Francesco and colleagues found a high rate of discrepancy. Of 42 clarithromycin-resistant strains, only 23 harbored the 3 known mutations, whereas 19 did not. These investigators identified the following mutations in 14 of 19 cases: A2115G, G2141A, and A2144T.
Confirmation of such findings has not yet been made. In our recent experience (2014), there were only 3 discrepancies between genotypic and phenotypic methods out of 400 strains tested (Mégraud, 2015).
Others mechanisms that could be involved in clarithromycin resistance concern efflux pumps, as was found in campylobacters.
Methods
There are various molecular methods to detect these mutations, essentially polymerase chain reaction (PCR)-based methods as well as a non-PCR-based method, fluorescent in situ hybridization (FISH).
One of the most efficient methods is real-time PCR. This method is described first. The other techniques are also reviewed.
Real-time polymerase chain reaction for detection of Helicobacter pylori resistance to macrolides
The beauty of this method is that it is first able to detect the presence of H pylori with a better sensitivity than other methods, including culture. In our experience, 3% to 5% of true positives detected by PCR cannot be cultured probably because of preanalytical problems.
Principle
The first step consists of designing primers specific for H pylori on the 23S rRNA gene used as the target gene, on both sides of the mutation site. The second step is to design probes inside the fragment to be amplified: a 3′ anchor probe labeled with a fluorochrome (eg, fluorescein) and a 5′ sensor probe labeled with another fluorochrome (eg, LC-Red640), which must be located close to the other (3 bases upstream) to allow an energy transfer from the former to the latter. This is the principle of so-called fluorescence resonance energy transfer (FRET), performed in a LightCycler thermocycler (Roche Diagnostics, Neuilly sur Seine, France), which allows the amplicon formation to be followed in real time.
If the 23S rRNA gene of H pylori is present in the mixture, a curve is obtained after 35 cycles, allowing the identification of an H pylori –positive specimen.
Then, a melting curve analysis (MCA) is performed (ie, the temperature of the mixture is increased to determine the temperature at which dissociation of the double amplicon strands occurs). In the case of a wild-type, the melting temperature is the highest (62°C), whereas in the case of a mismatch, the melting temperature is lower: 58°C for the transversion and 53°C to 54°C for the transition ( Fig. 2 ). This approach was also performed by Matsumura and colleagues with different primers and probes.
Historically, this method was first proposed using SYBR green, a fluorophore specific for double-stranded DNA and used as a quencher, which transfers its energy to a second fluorophore Cy5 fixed on a probe specific to H pylori .
Advantages of this procedure
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It is not necessary to have viable organisms, so the transport conditions are not as strict as for culture, because DNA remains unaltered even at ambient temperature during long periods.
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The procedure can be performed within a few hours. DNA must be extracted: this can be done in various ways, including the use of commercial kits, and it can also be automatized. Then, the amplification reaction is performed within 2 hours.
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Mixed populations made of resistant and susceptible bacteria can be better detected than by traditional-based culture methods.
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There are commercially available PCR kits that offer a standardized procedure: ClariRes Assay (Ingenetix, Vienna, Austria), MutaReal (Immundiagnostic, Bensheim, Germany). Another interesting point is that this methodology can be applied not only to fresh biopsy specimens but also to archival material (eg, fixed material on histologic preparations), as well as on other specimens in which culture is seldom positive (ie, stool specimens). However, the accuracy of the results of H pylori detection in stools is still controversial. The amount of H pylori DNA in stools is not important, and inhibitors of the Taq polymerase may decrease the sensitivity of the method unless a long DNA extraction procedure is performed.
A variation of the method proposed by Lascols and colleagues consists of a quantitative detection of H pylori in gastric biopsy specimens followed, in the event of a positive result, by the performance of another hybridization with a different biprobe, followed by an MCA.
Variants of the method
The TaqMan format
For each of the 3 known mutations (A2142C, A2142G, and A2143G) and the wild-type of the 23S rRNA gene, 4 TaqMan-MGB (Minor Groove Binder) probes are designed. A first probe having the fluorochrome VIC allows the detection of the wild-type, a second labeled with fluorochrome FAM detects the mutated form A2142C, a third FAM probe the A2142G mutation, and a fourth FAM probe the A2143G mutation. It is necessary to perform several amplifications for each specimen and test the corresponding probes.
The fluorescence emitted by the activated fluorochrome hydrolyzed probe is then detected.
Advantages and limitations
The absence of melting curve requires different amplifications to be performed for each sample and the use of 4 TaqMan probes, which increases the cost.
The scorpion format
An alternative method described by Burucoa and colleagues is based on a single-vessel multiplex real-time PCR that detects H pylori infection and the wild-type sequence and the 3 mutations conferring clarithromycin resistance using allele-specific scorpion primers directly on biopsy specimens. The scorpion primers combine a primer and a probe in a single molecule and are able to distinguish between single nucleotide polymorphisms. Fluorescent signals produced when the probes are annealed are read in 4 channels by a SmartCycler thermocycler (Cepheid, Sunnyval, CA, USA).
Multiplex polymerase chain reaction followed by strip hybridization
The principle is the same as for real-time PCR (ie, the mixture contains primers to specifically amplify H pylori and others targeting the 23S rRNA gene). Once the PCR is carried out, there is a second step of DNA strip hybridization. Strips coated with different oligonucleotides (DNA probes) are commercially available. The probes are designed to hybridize with the sequences of the wild-type alleles or the mutated alleles.
To assess positive and negative bands, the strips are pasted to an evaluation sheet after hybridization, and a template is aligned with the conjugate control band of the respective strip. Control bands of the conjugate control and amplification control should appear positive ( Fig. 3 ). Using the commercially available GenoType HelicoDR (Hain LifeScience, Nehren, Germany) a concordance score of 0.96 was found with real-time PCR for clarithromycin resistance. The sensitivity and specificity in the published studies are presented in Table 1 .
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