Digital PCR to Detect and Quantify Heteroresistance in Drug Resistant Mycobacterium tuberculosis

Abstract

Drug resistance in Mycobacterium tuberculosis presents an enormous public health threat. It is typically defined as >1% of drug resistant colonies using the agar proportion method. Detecting small numbers of drug resistant Tb in a population, also known as heteroresistance, is challenging with current methodologies. Here we have utilized digital PCR to detect heteroresistance within M. tuberculosis populations with excellent accuracy versus the agar proportion method. We designed dual TaqMan-MGB probes to detect wild-type and mutant sequences of katG (315), rpoB (531), gyrA (94,95) and rrs (1401), genes that associate with resistance to isoniazid, rifampin, fluoroquinolone, and aminoglycoside respectively. We generated heteroresistant mixtures of susceptible and extensively drug resistant Tb, followed by DNA extraction and digital PCR. Digital PCR yielded a close approximation to agar proportion's percentages of resistant colonies, and yielded 100% concordance with agar proportion's susceptible/resistant results. Indeed, the digital PCR method was able to identify mutant sequence in mixtures containing as little as 1000∶1 susceptible:resistant Tb. By contrast, real-time PCR or PCR followed by Sanger sequencing were less sensitive and had little resolution to detect heteroresistance, requiring fully 1∶1 or 10∶1 susceptible:resistant ratios in order to detect resistance. Our assay can also work in sputum so long as sufficient quantities of Tb are present (>1000 cfu/ml). This work demonstrates the utility of digital PCR to detect and quantify heteroresistance in drug resistant Tb, which may be useful to inform treatment decisions faster than agar proportion.

Digital PCR for Detection and Quantification of Fluoroquinolone Resistance in Legionella pneumophila

“…Consequently, the Sanger sequencing and qPCR tests may not be able to detect antibiotic-resistant subpopulations of L. pneumophila in the early stage of infection in legionellosis patients. The dPCRgyrA sensitivity levels observed in this study are similar to those previously reported by Pholwat et (23). In the literature, the maximum sensitivity reported for dPCR is 0.001% (one mutant sequence for 100,000 wild-type sequences) (28)(29)(30).…”

“…dPCR has wide clinical applications in the oncology field (14) and ongoing applications in noninvasive prenatal diagnosis (15,16) and organ transplant rejection monitoring (17). For infectious diseases, dPCR technology has been mainly used for the diagnosis of viral diseases (13), the quantification of bacteria in clinical samples (18)(19)(20)(21), the detection and quantification of antibiotic resistance genes (22,23), and the detection and quantification of bacterial toxins (24).…”

PhyResSE: a Web Tool Delineating Mycobacterium tuberculosis Antibiotic Resistance and Lineage from Whole-Genome Sequencing Data

“…For example, one isolate harbored an rpoB resistance-mediating mutation (CCG [Pro] at codon 452) in 19% of the total population, as deduced from our WGS data, which is below the limit of detection by Sanger sequencing (Fig. 4) (33). One discordant SNP is covered by the reverse primer used for Sanger sequencing, which leads to a wild-type call (data not shown).…”

Detection of Heteroresistant Mycobacterium tuberculosis by Pyrosequencing

“…Thus, NGS could potentially offer a better understanding of the population structure in clinical samples. Digital PCR may be another alternative to detect heteroresistance more precisely (18).…”