Dataset Information

Automatic Identification of Individual rpoB Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay.

ABSTRACT: Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the rpoB gene by the use of melting temperature (Tm ) information from 4 rpoB probes which can fall in one of the 9 different assay-specified Tm windows. The large amount of Tm data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the rpoB RRDR by utilizing the pattern of the Tm of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific "Tm signature" reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the Tm distances between the test sample and the reference Tm values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of rpoB single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.

SUBMITTER: Cao Y

PROVIDER: S-EPMC6935902 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Publications

Automatic Identification of Individual rpoB Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay.

Cao Yuan Y Parmar Heta H Simmons Ann Marie AM Kale Devika D Tong Kristy K Lieu Deanna D Persing David D Kwiatkowski Robert R Alland David D Chakravorty Soumitesh S

Journal of clinical microbiology 20191223 1

Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the rpoB gene by the use of melting temperature (Tm ) information from 4 rpoB probes which can fall in one of the 9 different assay-specified Tm windows. The large amount of Tm data generated by t ...[more]

PMID: 31533985

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Project description:BackgroundXpert MTB/RIF Ultra (Xpert Ultra) might have higher sensitivity than its predecessor, Xpert MTB/RIF (Xpert), but its role in tuberculous meningitis diagnosis is uncertain. We aimed to compare Xpert Ultra with Xpert for the diagnosis of tuberculous meningitis in HIV-uninfected and HIV-infected adults.MethodsIn this prospective, randomised, diagnostic accuracy study, adults (≥16 years) with suspected tuberculous meningitis from a single centre in Vietnam were randomly assigned to cerebrospinal fluid testing by either Xpert Ultra or Xpert at baseline and, if treated for tuberculous meningitis, after 3-4 weeks of treatment. Test performance (sensitivity, specificity, and positive and negative predictive values) was calculated for Xpert Ultra and Xpert and compared against clinical and mycobacterial culture reference standards. Analyses were done for all patients and by HIV status.FindingsBetween Oct 16, 2017, and Feb 10, 2019, 205 patients were randomly assigned to Xpert Ultra (n=103) or Xpert (n=102). The sensitivities of Xpert Ultra and Xpert for tuberculous meningitis diagnosis against a reference standard of definite, probable, and possible tuberculous meningitis were 47·2% (95% CI 34·4-60·3; 25 of 53 patients) for Xpert Ultra and 39·6% (27·6-53·1; 21 of 53) for Xpert (p=0·56); specificities were 100·0% (95% CI 92·0-100·0; 44 of 44) and 100·0% (92·6-100·0; 48 of 48), respectively. In HIV-negative patients, the sensitivity of Xpert Ultra was 38·9% (24·8-55·1; 14 of 36) versus 22·9% (12·1-39·0; eight of 35) by Xpert (p=0·23). In HIV co-infected patients, the sensitivities were 64·3% (38·8-83·7; nine of 14) for Xpert Ultra and 76·9% (49·7-91·8; ten of 13) for Xpert (p=0·77). Negative predictive values were 61·1% (49·6-71·5) for Xpert Ultra and 60·0% (49·0-70·0) for Xpert. Against a reference standard of mycobacterial culture, sensitivities were 90·9% (72·2-97·5; 20 of 22 patients) for Xpert Ultra and 81·8% (61·5-92·7; 18 of 22) for Xpert (p=0·66); specificities were 93·9% (85·4-97·6; 62 of 66) and 96·9% (89·5-91·2; 63 of 65), respectively. Six (22%) of 27 patients had a positive test by Xpert Ultra after 4 weeks of treatment versus two (9%) of 22 patients by Xpert.InterpretationXpert Ultra was not statistically superior to Xpert for the diagnosis of tuberculous meningitis in HIV-uninfected and HIV-infected adults. A negative Xpert Ultra or Xpert test does not rule out tuberculous meningitis. New diagnostic strategies are urgently required.FundingWellcome Trust and the Foundation for Innovative New Diagnostics.

Project description:The aim of this study was to evaluate the performance of Xpert MTB/RIF Ultra (Ultra) compared with its predecessor, Xpert MTB/RIF (Xpert), in the diagnosis of tuberculosis (TB) in a low TB incidence country. Retrospective analysis was performed on 689 clinical samples received between 2015 and 2018, on which Xpert was performed, and on 715 samples, received between 2018 and 2020, on which Ultra was performed. Samples were pulmonary (n = 830) and extrapulmonary (n = 574) in nature, and a total of 264 were culture positive for Mycobacterium tuberculosis complex (MTBC). The diagnostic performance of both assays was analyzed using culture as the reference standard. The sensitivity of Ultra for culture positive (smear positive and smear negative) MTBC samples, was 93.2% (110/118) compared with 82.2% (120/146) for Xpert (P = 0.0078). In smear negative-culture positive samples, Ultra had a sensitivity of 74.2% (23/31) versus 36.11% (13/36) for Xpert (P = 0.0018). Specificity of both assays was comparable at 94.8% (566/597) for Ultra and 95.8% (520/543) for Xpert (P = 0.4475). The sensitivity of Ultra and Xpert assays among exclusively pulmonary samples was 95.3% (82/86) and 90.3% (84/93), respectively (P = 0.1955), and 87.5% (28/32) and 67.9% (36/53), respectively, among extrapulmonary samples (P = 0.0426). Ultra showed improved performance compared with Xpert in a low TB incidence setting, particularly in smear negative and extrapulmonary MTBC disease. The specificity of Ultra was lower than Xpert, however, this was not statistically significant. IMPORTANCE The study demonstrates the improved sensitivity of the Ultra compared with the Xpert, particularly in smear negative TB disease, for both pulmonary and extrapulmonary samples in a low TB incidence setting. Cycle threshold (Ct) value for both assays was found to positively correlate with time to TB culture positivity, suggesting that Ct and semiquantitative results could be used as indicators of sample MTBC bacillary burden, and thus, perhaps, of transmission potential. This may have implications for the designation of patient isolation precautions.

Dataset Information

Automatic Identification of Individual rpoB Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay.

Publications

Automatic Identification of Individual <i>rpoB</i> Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay.

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