High-Level Primary Clarithromycin Resistance of Helicobacter pylori in Algiers,Algeria: A Prospective Multicenter Molecular Study

Introduction

Helicobacter pylori infection is one of the most common chronic bacterial infections in the world and has been established as a major cause of gastritis, peptic ulcer disease, and gastric cancer.^8,9,19 Clarithromycin is a key component of the standard triple therapy used in the eradication of H. pylori. However, clarithromycin resistance has been increasing in many countries and constitutes the main risk factor for treatment failure, reducing the efficacy of first-line therapy by up to 70%.^10,12,16 Clarithromycin resistance is due to point mutation in the 23S ribosomal RNA (rRNA) gene. Three mutations in the rRNA gene have been described: A2142G, A2143G, and A2142C.^13,21 Although other mutations in the 23S rRNA gene have also been suspected of conferring resistance, their role is not yet confirmed. ⁴

Resistance of H. pylori to clarithromycin can be detected either by phenotypic susceptibility testing or by molecular techniques based on PCR.^5,13 Phenotypic susceptibility testing requires the isolation of the bacterium cultured from gastric biopsy. Culture is difficult to achieve and available only in a few specialized laboratories. The molecular techniques aimed at detecting mutations that confer resistance to clarithromycin are easier to perform, faster and more efficient than phenotypic susceptibility testing.^3,6 In 2008, we developed a quadruplex real-time PCR assay using Scorpion primers with good performances (Se 0.98, Sp 0.98). ³ The Scorpion PCR has been used in several molecular studies in France, Tunisia, Senegal, Iran, and Morocco.^1,2,15,16,18

The Maastricht IV consensus report for the management of H. pylori infection indicates that the threshold of clarithromycin resistance at which the standard clarithromycin-based triple therapy should not be used or a clarithromycin susceptibility test should be performed is 15–20%. ⁸ However, in many countries, the current prevalence of resistance remains unknown. In Algeria, the prevalence of H. pylori infection in adults was estimated in 1987 as ranging from 80% to 90%, but the prevalence of H. pylori resistance to clarithromycin is still unknown.^11,20

The aim of this study was to evaluate, for the first time in Algeria, the prevalence of primary resistance of H. pylori to clarithromycin among patients never previously treated for H. pylori infection.

Materials and Methods

Results

A total of 195 Algerian patients referred for gastroduodenal endoscopy were recruited in Algiers: 115 in the Mustapha Bacha University Hospital, 42 in the Bologhine General Hospital, 24 in the Bab El Oued University Hospital, and 14 among private gastroenterologists in Algiers. There were 75 men and 120 women aged from 13 to 85 years, with a mean age of 46.6 years. Sex ratio and mean age were not statistically different in the four inclusion centers (Table 1). Fourteen patients had previously received at least one treatment against H. pylori infection. Endoscopic examination detected 14 patients with a duodenal or gastric ulcer. The Scorpion PCR detected H. pylori DNA in 91 biopsies (47%) (Table 2). All negative Scorpion PCR extracts were positive for β-hemoglobin gene PCR detection, thereby confirming the absence of PCR inhibitors.

A mutation conferring resistance to clarithromycin was detected in 32 of the 91 positive samples (35%) and in 29 of the 88 patients never previously treated for an H. pylori infection (33%) (Table 2). The prevalence of primary resistance of H. pylori to clarithromycin in this Algerian population was 33%. Women were more frequently infected with a resistant strain than men (48.3% vs. 24.2%, but without statistical significance, p = 0.099). Among the 32 biopsies detected positive for resistance by Scorpion PCR, 26 (81%) harbored a A2143G mutation, 3 (9.4%) a A2142G mutation, and 2 (6.2%) a A2142C mutation, with one biopsy harboring a mixture of both A2142G and A2142C mutations. In 9 (28%) biopsies, Scorpion PCR revealed a mixed infection (detection of a susceptible and a resistant strain).

Discussion

The Scorpion PCR detected H. pylori DNA in 91 biopsies (47%) (Table 2). This prevalence of H. pylori infection among patients referred for gastroduodenal endoscopy appears relatively low for a developing country, especially when compared to the result of a 1987 study, which reported a seroprevalence of 80% in the Algerian population.^11,20 Serological studies cannot determine the prevalence of active H. pylori infections because the antibodies can persist for many years after the successful eradication therapy, whereas PCR will only detect active infections. This unexpectedly low prevalence may reflect the global decline in the prevalence of H. pylori infection. This global decrease of H. pylori infection has already been described in different countries and is believed to be due to improved hygiene and actively contributing elimination by antibiotics during childhood, both of them contributing to declining transmission risk. ⁷ A decrease of almost 30% in 30 years of the prevalence of H. pylori infection as we have observed in Algeria between the 1987 seroprevalence study and our study is similar to that of 26% in 22 years reported in Finland. ¹⁷

The Scorpion PCR we used to detect H. pylori DNA and to determine primary clarithromycin resistance was validated by excellent performances (Se 0.98, Sp 0.98) when tested on 259 biopsy specimens using culture and/or RFLP-PCR as a gold standard. ³ Since this validation, this method has been used in different collaborative studies published with our team and also by another team.^1,2,15,16,18 Inclusion conditions (consecutive patients but restricted to periods where we could dispose of reagents for DNA extraction) were not identified as putative bias to explain this unexpected low prevalence of H. pylori infection. There was no refusal to participate in this study. We can only suspect recruitment bias favoring the inclusion of a population with higher living standards than those of the general population in Algeria. Low socioeconomic level is a recognized risk factor for H. pylori infection.^7,9 The central location, in the capital city, of inclusion centers could have promoted recruitment within an upper-middle class that is more well-off and better educated than the Algerian population of the countryside. However, the urbanization rate of Algeria exceeds 60%, diminishing the importance of this bias.

Knowledge of local antibiotic resistance rate is crucial to the choice of an optimal empirical first-line treatment for the eradication of H. pylori infection. ⁸ The standard triple therapy containing clarithromycin proposed at the first Maastricht conference in 1997 has become universal as was recommended at all the consensus conferences held around the world. The loss of effectiveness found in many countries since the beginning of the 21st century is due to the increasing prevalence of clarithromycin resistance. ¹⁰ The most recent Maastricht IV consensus recommends that “PPI-Clarithromycin-containing triple therapy without prior susceptibility testing should be abandoned when the clarithromycin resistance rate in the region is more than 15–20%.” ⁸ In Algeria, as in many countries, the clarithromycin-based empirical triple therapy is widely used as a first-line eradication treatment, but the primary clarithromycin resistance rate remains unknown. We are the first to report in Algeria a resistance rate reaching 33%, largely over the 15–20% threshold put forward by the Maastricht IV consensus report as a reason to abandon clarithromycin-based first-line treatment. Two possibilities can be proposed to Algerian clinicians for the treatment of H. pylori-infected patients: either a new regimen involving a combination of more antibiotics or a new therapeutic strategy guided by antimicrobial susceptibility testing. In areas of high clarithromycin resistance, bismuth-containing quadruple treatments are recommended. ⁸ In Algeria, however, bismuth is not available. As a result, concomitant or sequential treatments are recommended despite the obvious ecological consequences on gastroenterological microflora and adverse effects. A recent analysis indicated that 14-day concomitant therapy should be preferred because it is the most effective way of overcoming dual resistance (clarithromycin and metronidazole resistance). ¹⁴ Although we do not provide information in this study about metronidazole resistance, metronidazole resistance rates are predictably high in Algeria, as is the case in many other African countries.^1,18

The alternative is to use a treatment guided by the results of resistance detection. Several studies have shown the benefit of a phenotypic resistance-guided therapeutic strategy. ²² Difficulty in obtaining the phenotypic susceptibility of H. pylori renders this strategy difficult to apply on a large scale in a developing country. Molecular techniques that are easier to implement, quicker and more effective than culture, are an interesting alternative that would facilitate the establishment of a therapeutic strategy guided by the results of molecular tests. As an example, the culture of H. pylori has been implemented since 2008 in the laboratory of the CHU Mustapha Bacha but with only limited performance, a shortcoming that motivated the development of molecular techniques.

The 33% clarithromycin resistance level reported in this study is relatively high compared to the resistance rates observed around the world. In Maghreb, only two published studies have reported clarithromycin resistance: a study conducted from 2005 to 2007 in Tunisia reported the prevalence of primary resistance to clarithromycin reaching 15.4%, whereas a recent study conducted from 2011 to 2014 in Morocco reported a prevalence reaching 28.2%.^1,2 Across the Mediterranean, similar clarithromycin resistance rates have been observed in several Southern European countries (Italy 26.7% and Portugal 31.5%). ¹² In Europe, a significant positive association between outpatient antibiotic usage and the level of primary resistance observed in H. pylori to key antimicrobial agents has been demonstrated, particularly for primary clarithromycin resistance level and macrolide consumption. ¹² In the absence of accurate knowledge on antibiotic consumption in Algeria, we can only suspect a high consumption of macrolides.

We report a distribution of mutations with a predominance of the A2143G mutation; interestingly enough, A2142G predominates in Morocco, whereas A2143G predominates in France and Tunisia.^1,2,16

The high level of clarithromycin reported in this study, which highlights the need for regular surveillance of growing resistance and has a strong impact on clinical practice, still needs to be confirmed by larger multicenter studies in Algeria and in Maghreb.

In conclusion, we report a high level of primary clarithromycin resistance in the Algerian population (33%), which leads us to recommend the abandonment of standard clarithromycin-based triple therapy as a first-line treatment in Algeria.