Updating the approaches to define susceptibility and resistance to anti-tuberculosis agents: implications for diagnosis and treatment

11 páginas, 2 figuras, 1 tablaInappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance.I. Comas was supported by PID2019-104477RB-I00 from the Spanish Science Ministry and by ERC (CoG 101001038)Peer reviewe

Digital PCR to detect and quantify heteroresistance in drug resistant Mycobacterium tuberculosis.

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

Rapid First- and Second-Line Drug Susceptibility Assay for Mycobacterium tuberculosis Isolates by Use of Quantitative PCR▿

The slow turnaround time for Mycobacterium tuberculosis drug susceptibility results is a barrier to care. We developed a rapid quantitative PCR (qPCR)-based phenotypic antimicrobial susceptibility test that utilizes amplification of the M. tuberculosis 16S rRNA gene after 3 days of incubation with antituberculosis drugs. To decrease background from killed organisms, we used propidium monoazide (PMA), a DNA-binding dye that penetrates damaged bacterial cells and renders DNA unamplifiable. M. tuberculosis was cultured in broth media containing PMA with or without drugs for 3 days prior to DNA extraction and real-time PCR amplification. 16S rRNA qPCR exhibited a significant decrease in threshold cycle (CT) time values (CT control − CT drug treated) with drug-susceptible strains compared with resistant strains. Susceptibility data were reported as ΔCT or as 2ΔCT and with appropriate cutoffs yielded an accuracy of 89 to 100% on 38 susceptible, multidrug-resistant, and extensively drug-resistant strains compared with conventional agar proportion susceptibility results for isoniazid, rifampin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide, para-aminosalicylic acid, linezolid, and cycloserine and compared with Bactec MGIT results for pyrazinamide. This PMA-qPCR assay is useful as a rapid 3-day first- and second-line drug susceptibility test for M. tuberculosis

Real-time PCR of undiluted DNA.

<p>The undiluted DNA of each H37Rv:XDR-TB mixture underwent real-time PCR for each gene with both probes. <i>katG</i> is shown, with detection of mutant sequence (blue trace) at H37Rv:XDR-TB mixtures of 0∶1, 1∶1, and late detection at 10∶1. Detection of wild-type sequence occurred at all H37Rv containing mixtures. qPCR thresholds were set per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057238#pone-0057238-g001" target="_blank">Figure 1</a> (red and blue horizontal lines for wild-type and mutant fluorophores, respectively). Similar results were found for other genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057238#pone.0057238.s001" target="_blank">Figure S1</a> for <i>rpoB</i>, <i>gyrA</i>, <i>rrs</i>).</p

Comparison of digital PCR, agar proportion, real-time PCR, and PCR with Sanger sequencing.

<p>R = resistant. S = susceptible. Agar proportion resistance defined as >1% resistant colonies, and the actual percentage of resistant colonies is shown in parentheses. Digital PCR resistance defined as >1% of PCR reactions indicating mutant sequence, with the actual percentage of PCR reactions with mutant sequence shown in parentheses. Real-time PCR resistance defined as mixed populations of wild-type and mutant sequence. Sequencing defined resistance as evidence of mutant sequence by chromatogram.</p

Digital PCR.

a<p>% mutant = [(n mixed population/2)+n mutant]/total n amplified.</p>b<p>Since the <i>rpoB</i> wild-type probe showed substantial cross reactivity to mutant target (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057238#pone-0057238-g001" target="_blank">Fig.1B</a>), detection of both wild-type and mutant <i>rpoB</i> probes in a single well was interpreted as presence of mutant sequence if fluorescence signal of mutant probe was higher than that of the wild-type probe, and interpreted as a mixed population if fluorescence signal of the mutant probe was equal or lower than that of the wild-type probe. Detection of wild-type only <i>rpoB</i> was interpreted as wild-type sequence.</p

Digital PCR of spiked sputum samples using <i>rpoB</i>^a primers and probes.

a<p>Since the <i>rpoB</i> wild-type probe showed substantial cross reactivity to mutant target (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057238#pone-0057238-g001" target="_blank">Fig.1B</a>), detection of both wild-type and mutant <i>rpoB</i> probes in a single well was interpreted as presence of mutant sequence if fluorescence signal of mutant probe higher than that of the wild-type probe, and interpreted as a mixed population if fluorescence signal of the mutant probe was equal or lower than that of wild-type probe. Detection of wild-type only <i>rpoB</i> was interpreted as wild-type sequence.</p>b<p>the dilution that yielded approximately 50% PCR positive.</p>c<p>% mutant = [(n mixed population/2)+n mutant]/total n amplified.</p

Digital PCR.

<p>The DNA of each H37Rv:XDR-TB mixture was diluted 1∶10000 and subjected to real-time PCR for each gene with both probes in 384 well plates. This figure shows representative 10∶1 (<i>katG, gyrA, rrs</i>) or 100∶1 (<i>rpoB</i>) mixtures. Individual wells revealed detection with the wild-type probe (red traces), mutant probe (blue traces), both, or neither. qPCR thresholds were set per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057238#pone-0057238-g001" target="_blank">Figure 1</a> (red and blue horizontal lines for wild-type and mutant fluorophores, respectively). Cycling continued for 55 cycles (for <i>katG</i>) due to dilute DNA template.</p

Determination of dilution for digital PCR.

<p>The DNA of each H37Rv:XDR-TB mixture was diluted 10 fold serially and subjected to real-time PCR using the <i>rpoB</i> primers and probes. The percent of PCR reactions that amplified at each dilution is shown. The target one-half genome equivalent DNA is the amount of DNA that yielded 50% positive <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057238#pone.0057238-Vogelstein1" target="_blank">[10]</a>.</p