A two-step PCR-based assay of a small region of the Helicobacter pylori (H. pylori) bacteria can help detect H. pylori infection and also identify clarithromycin-resistant bacteria and those that are drug-sensitive in six-seven hours has been developed by a team of researchers from the National Institute of Cholera and Enteric Diseases (ICMR-NICED), Kolkata. Since H. pylori bacteria grow slowly, it takes about a week to culture the bacteria and a couple of more weeks to test for drug-sensitivity, which the new diagnostic assay bypasses. The molecular-based assay has been found to have 100% sensitivity and specificity.
There is an increasing trend of clarithromycin-resistant H. pylori bacteria in India leading to a decreasing success rate in treating the infection.
Most of the infections caused by the bacterium H. pylori are asymptomatic, 10–15% of them develop peptic ulcer disorders or stomach cancer. In India, H. pylori infections affect 60-70% of the population. H. pylori infection is often acquired during childhood and remains in the stomach throughout life if not treated with antibiotics effectively. So, if someone suffers from gastroduodenal diseases along with the detection of H. pylori infection, eradication of the bacteria provides the most effective treatment. Importantly, H. pylori infection is one of the robust known risk factors for gastric cancer.
As it takes three-four weeks to culture the bacteria and carry out drug-sensitivity tests, drug-resistant studies of H. pylori are seldom carried out in India. So, the conventionally used empirical treatment using clarithromycin is routinely used without knowing the drug-sensitivity. The growing incidence of clarithromycin-resistant bacteria is a big concern and has to be addressed as it is the most important reason for treatment failure. The NICED researchers undertook the study to identify the root cause of resistance toward clarithromycin and to develop a molecular-based technique for rapidly detecting antibiotic resistance.
The team led by Dr. Asish Kumar Mukhopadhyay from NICED turned to genome sequencing to identify that the drug resistance was due to a point mutation (A to G mutation at 2143 position) in the 23S ribosomal RNA (rRNA) gene of the bacteria. To confirm that the point mutation was indeed responsible for drug-resistance, the researchers isolated and amplified 617 base pairs that contained the point mutation and transferred the base pairs to drug-sensitive bacteria. “The drug-sensitive bacteria carrying the point mutation became resistant to the drug thus confirming that the point mutation was indeed responsible for developing resistance towards clarithromycin,” Dr. Mukhopadhyay tells The Hindu.
To further confirm the role of point mutation in drug resistance, the team sequenced the bacteria that had become drug-resistant after the base pairs were transferred and found that the point mutation was present in the bacteria. The results were published recently in the journal Gut Pathogens.
Bioinformatics study revealed that drug-resistant and drug-sensitive strains had very different binding affinity for the drug — the drug’s binding affinity to the mutant was weaker compared with drug-sensitive bacteria. “Due to weak binding, less amount of the drug is able to get into the bacteria, and so is unable to kill them. The point mutation is thus responsible for the clarithromycin resistance,” he says.
The DNA template used for assay was prepared by amplifying a small segment containing the point mutation from bacteria isolated directly from biopsy samples. The DNA template prepared from bacteria isolated directly from biopsy samples was validated with the DNA template prepared from bacteria that was cultured. “This was done to confirm the validity of the DNA template prepared from bacteria taken from biopsy samples,” says Dr. Mukhopadhyay. The DNA template was used for the PCR-based assay.
The researchers developed a two-step PCR-based assay to first detect H. pylori infection and then to differentiate resistant isolates from sensitive ones directly from biopsy samples. In the initial step of PCR, the 617 base-pair segment containing the point mutation was amplified using DNA templates isolated from biopsy samples. In the second PCR step, 183 base pairs amplified by the first PCR step are used as a template. For the second PCR step, two allele-specific primer sets have been designed by exploiting the point mutation in the resistant strains. “The clarithromycin-resistant strains will get amplified only by the resistant-specific primer and not with the sensitive-specific primer. Similarly, the sensitive strains will get amplified only by the sensitive-specific primer and not the resistant-specific primer,” he explains.
“The two-steps PCR method was evaluated by comparing it with the conventional drug sensitivity method and also by sequencing analysis, which showed 100% sensitivity and specificity,” says Dr. Mukhopadhyay.
