Xpert Ultra can unambiguously identify specific rifampicin resistance-conferring mutations

Abstract

Phone: +32(0)33455345 21 Fax number: +32(0)32476333 22 23 24 2 Introduction 25The deluge of data produced by XpertMTB/RIF (Cepheid) can help improve global 26 rifampicin-resistant tuberculosis (RR-TB) control strategies through molecular 27 epidemiological surveillance (1, 2). Recently, a new version of the test -Xpert Ultra 28 (hereinafter called Ultra) was released (3). Determining the relationship between RR-29 conferring rpoB mutations, Ultra probes, and melting temperature shifts (∆Tm)the 30 difference between mutant and wildtype melting temperaturesallows Ultra results to 31 be utilized for rapid detection of RR-TB strains and related underlying rpoB mutations. 32 Methods 33To validate the usefulness of Ultra results for predicting specific mutations, we analyzed 34 10 RS-TB and 107 RR-TB strains from the Belgian Coordinated Collections of 35Microorganisms 36 in the Institute of Tropical Medicine, Antwerp, Belgium. These strains harbor 36 unique 37 RR-conferring mutations determined by rpoB sequencing. 38 Results 39Overall, 31/32 (97%) mutations inside the Rifampicin Resistance Determining Region 40 (RRDR) were correctly identified by Ultra. Of concern, mutation His445Arg gave a "RIF 41Resistance INDERTERMINATE" result among 3/4 strains tested while it was reported as 42 RR in the initial validation study (3). The silent mutation Thr444Thr was not reported as 43 RR (Figure 1). The RR-conferring mutations on codons 170, 250, 299, 482, and 491 44 situated outside the RRDR were not detected. 45The probe reactions observed were largely in agreement with previous results (3) albeit 46 we noted that mutations Met434Val, Met434Thr and those in codon 435 were captured 47 only by probe rpoB2; Ser450Leu and Ser450Trp were captured by both probes rpoB3 48 3 and rpoB4a, His445Arg was captured only by probe rpoB3; and Lys446Gln was 49 captured only by probe rpoB4. 50All mutations except those in codon 450 were associated with a negative ∆Tm (Figure 51 2). The combination of ∆Tm values with the capturing probes enabled to differentiate 52 mutations in codons 428, 430, 431, 432, 434, 435, 441, 445, 446, and 452, including 53 disputed mutations (4) ( Table 1). Mutation Asp435Tyr was unambiguously distinguished 54 from Asp435Val through probe rpoB2|∆Tm|, while mutations Ser441Gln and Ser441Leu 55 were discriminated from the rest by|∆Tm| values of probes rpoB2 and rpoB3. Mutations 56 His445Asp and His445Tyr were distinguished from disputed mutations His445Leu and 57 His445Asn through probe rpoB3 |∆Tm|. Ser450Leu was distinguished from Ser450Trp 58 by probe rpoB4A |∆Tm| except for one strain with an outlier rpoB4A Tm of 70.9°C in 59 contrast to the other 13 strains with rpoB4A Tm of 73.3-73.8°C. The indeterminate result 60 associated with His445Arg may be caused by its |∆Tm|=1.8°C compared with |∆Tm| 61 typically exceeding 2°C for other mutations. 62 Conclusions 63Our findings confirm the ability of Ultra to unambiguously identify a wide range of RRDR 64 mutations. With the unprecedented roll-out of XpertMTB/RIF and asso...