Ng, K.C.S.

Bridging diagnostic gaps may help reduce the global burden of rifampicin-resistant tuberculosis (RR-TB) transmission. Inaccurate diagnoses result in false RR-TB patients receiving toxic and less effective drugs, while false rifampicin-susceptible-TB patients continue to transmit RR-TB in the community which may lead to RR-TB outbreaks. Early detection of RR-TB outbreaks entails rapid and systematic analysis of RR data at the population level. The increasing use of Xpert MTB/RIF as a frontline diagnostic test for all presumptive TB patients provides an opportunity to utilize routinely produced RR-TB data for timely detection of RR-TB clusters. Currently, there is no system in place capable of performing this analysis.The PhD thesis of Kamela Charmaine S. Ng entitled “Utilization of molecular resistance test results as tools to support public health efforts for improved control of RR-TB” has improved the understanding of routine diagnostic data and provided policy implications on addressing diagnostic challenges that affect interruption of RR-TB transmission.Furthermore, this thesis developed an early warning tool which harnessed the full potential of routine Xpert data for improved RR-TB surveillance. This tool utilizes both projected site-specific numbers of RR-TB diagnoses and frequency of Xpert probe(s) which represents circulating RR-TB mutations in the setting. The National TB Control Program is then prompted to investigate further on Xpert site(s) with unusual RR-TB clusters, and tailor active case finding and initiate effective treatment to reduce the transmissibility of TB. This thesis demonstrates as a proof-of-concept, that routine Xpert data contain actionable information that could improve country surveillance of RR-TB

International Migration, Integration and Social Cohesion online publications

UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)UvA-DARE (Digital Academic Repository)Utilization of molecular resistance test results as tools to support public healthefforts for improved control of rifampicin-resistant tuberculosisNg, K.C.S.Publication date2020Document VersionOther versionLicenseOtherLink to publicationCitation for published version (APA):Ng, K. C. S. (2020). Utilization of molecular resistance test results as tools to support publichealth efforts for improved control of rifampicin-resistant tuberculosis. [Thesis, fully internal,Universiteit van Amsterdam].General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s)and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an opencontent license (like Creative Commons).Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, pleaselet the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the materialinaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letterto: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. Youwill be contacted as soon as possible.Download date:26 Oct 202363Chapter 3 Xpert Ultra can unambiguously identify specific rifampicin resistance-conferring mutationsKamela C. S. Ng,a Armand van Deun, a,b Conor J. Meehan,a Gabriela Torrea,a Michèle Driesen,a Siemon Gabriëls,a Leen Rigouts,a,c Emmanuel André,d* Bouke C. de Jonga* Unit of Mycobacteriology, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgiuma; International Union Against Tuberculosis and Lung Disease, Paris, France b; Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgiumc; Laboratory of Clinical Bacteriology and Mycology, KU Leuven, Leuven, Belgiumd* joint final authorsJournal of Clinical Microbiology 2018, 56:e00686-18. https://pubmed.ncbi.nlm.nih.gov/2992564364INTRODUCTIONThe deluge of data produced by XpertMTB/RIF (Cepheid) can help improve global rifampicin-resistant tuberculosis (RR-TB) control strategies through molecular epidemiological surveillance (1, 2). Recently, a new version of the test – Xpert Ultra (hereinafter called Ultra) was released (3). Determining the relationship between RR-conferring rpoB mutations, Ultra probes, and melting temperature shifts (∆Tm) – the difference between mutant and wildtype melting temperatures – allows Ultra results to be utilized for rapid detection of RR-TB strains and related underlying rpoB mutations. METHODSTo validate the usefulness of Ultra results for predicting specific mutations, we analyzed 10 RS-TB and 107 RR-TB strains from the Belgian Coordinated Collections of Microorganisms in the Institute of Tropical Medicine, Antwerp, Belgium. These strains harbor 36 unique RR-conferring mutations determined by rpoB sequencing.RESULTSOverall, 31/32 (97%) mutations inside the Rifampicin Resistance Determining Region (RRDR) were correctly identified by Ultra. Of concern, mutation His445Arg gave a “RIF Resistance INDERTERMINATE” result among 3/4 strains tested while it was reported as RR in the initial validation study (3). The silent mutation Thr444Thr was not reported as RR (Figure 1). The RR-conferring mutations on codons 170, 250, 299, 482, and 491 situated outside the RRDR were not detected.The probe reactions observed were largely in agreement with previous results (3) albeit we noted that mutations Met434Val, Met434Thr and those in codon 435 were captured only by probe rpoB2; Ser450Leu and Ser450Trp were captured by both probes rpoB3 and rpoB4a, His445Arg was captured only by probe rpoB3; and Lys446Gln was captured only by probe rpoB4.CHAPTER 365Figure 1. Overview of Xpert Ultra test results. The observed probe reactions for each RRDR mutation were laid over the claimed probe coverage (light gray). Shown in black are probe reactions concordant with manufacturer claims, in blue are probe reactions missed by one probe but captured by another probe, and in red is a probe reaction representing a “RIF Resistance Indeterminate” result from 3 out of 4 strains tested. Results in the hatched pattern were superimposed for greater visibility.All mutations except those in codon 450 were associated with a negative ∆Tm (Figure 2). The combination of ∆Tm values with the capturing probes enabled to differentiate mutations in codons 428, 430, 431, 432, 434, 435, 441, 445, 446, and 452, including disputed mutations (4) (Table 1). Mutation Asp435Tyr was unambiguously distinguished from Asp435Val through probe rpoB2|∆Tm|, while mutations Ser441Gln and Ser441Leu were discriminated from the rest by |∆Tm| values of probes rpoB2 and rpoB3. Mutations His445Asp and His445Tyr were distinguished from disputed mutations His445Leu and His445Asn through probe rpoB3 |∆Tm|. Ser450Leu was distinguished from Ser450Trp by probe rpoB4A |∆Tm| except for one strain with an outlier rpoB4A Tm of 70.9°C in contrast to the other 13 strains with rpoB4A Tm of 73.3-73.8°C. The indeterminate result associated with His445Arg may be caused by its |∆Tm|=1.8°C compared with |∆Tm| typically exceeding 2°C for other mutations. DISCRIMINATORY XPERT ULTRA DATA366Figure 2. Melting temperature shifts (ΔTms) observed upon detection of a rifampin resistance (RR)-conferring rpoB mutation in the RR-determining region (RRDR) by Xpert Ultra. The y axis reflects the melting temperature difference (ΔTm) between mutant and wild-type probe-amplicon hybrids, while the x axis shows the mutations that we tested. The data points on the graph are ΔTm values grouped by their associated Ultra probes (differentiated by color), which correspond to a specific rpoB mutation. x axis labels in brown are disputed mutations.CONCLUSIONSOur findings confirm the ability of Ultra to unambiguously identify a wide range of RRDR mutations. With the unprecedented roll-out of XpertMTB/RIF and associated connectivity solutions, such as DataToCare (Savics, Belgium) and GXAlert (SystemOne, USA) (2), Ultra results may be exploited to rule-out transmission between RR-TB patients in a specific setting, distinguish relapse from reinfection, and resolve discordance between an RR Ultra result and a low-level RS phenotypic result due to a disputed mutation. For such applications, it is key that ∆Tm values are included in the exported results.AcknowledgementThis work was supported by Erasmus Mundus Joint Doctorate Fellowship grant 2016-1346 to KCSN.CHAPTER 367Table 1.  Xpert Ultra raw resultsaaCapturing probes, wild-type melt peak temperature (Tm) ranges and means, mutant Tm ranges, and absolute values of melting temperature shift (ΔTm) ranges associated with specific rpoB mutations in the strains tested and the corresponding nucleotide changes. ND, strains that harbored corresponding mutations outside the RRDR yielded a “RIF Resistance Not Detected” result. *, rifampin resistance-determining region (RRDR) mutation unambiguously identified by unique combinations of Ultra probes and ΔTms, including disputed ones (in italics). #, rifampin susceptible according to phenotypic testing.bFor double mutants, the high-confidence RR-conferring mutations are underlined (6, 7). DISCRIMINATORY XPERT ULTRA DATA368REFERENCES1. Andre E, Isaacs C, Affolabi D, Alagna R, Brockmann D, de Jong BC, Cambau E, Churchyard G, Cohen T, Delmee M, Delvenne JC, Farhat M, Habib A, Holme P, Keshavjee S, Khan A, Lightfoot P, Moore D, Moreno Y, Mundade Y, Pai M, Patel S, Nyaruhirira AU, Rocha LE, Takle J, Trebucq A, Creswell J, Boehme C. 2016. Connectivity of diagnostic technologies: improving surveillance and accelerating tuberculosis elimination. Int J Tuberc Lung Dis 20:999-1003.2. Ng KC, Meehan CJ, Torrea G, Goeminne L, Diels M, Rigouts L, de Jong BC, Andre E. 2018. Potential Application of Digitally Linked Tuberculosis Diagnostics for Real-Time Surveillance of Drug-Resistant Tuberculosis Transmission: Validation and Analysis of Test Results. JMIR Med Inform 6:e12.3. Chakravorty S, Simmons AM, Rowneki M, Parmar H, Cao Y, Ryan J, Banada PP, Deshpande S, Shenai S, Gall A, Glass J, Krieswirth B, Schumacher SG, Nabeta P, Tukvadze N, Rodrigues C, Skrahina A, Tagliani E, Cirillo DM, Davidow A, Denkinger CM, Persing D, Kwiatkowski R, Jones M, Alland D. 2017. The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing. MBio 8.4. Van Deun A, Aung KJ, Bola V, Lebeke R, Hossain MA, de Rijk WB, Rigouts L, Gumusboga A, Torrea G, de Jong BC. 2013. Rifampin drug resistance tests for tuberculosis: challenging the gold standard. J Clin Microbiol 51:2633-40.CHAPTER 369SUPPLEMENTARY INFORMATIONSupplemental methods. Processing of rifampicin-resistant tuberculosis thermolysates for Xpert Ultratesting: methodological details.Figure S1. Interpreting Xpert Ultra ΔTm plus probe reactions observed in 2 rifampin-resistant tuberculosis patients for evaluation of a potential transmission event.FigureS2. Interpreting Xpert Ultra ΔTm plus probe reactions observed in 2 rifampin-resistant tuberculosis patients for identification of relapse cases.https://jcm.asm.org/highwire/filestream/123403/field_highwire_adjunct_files/0/zjm999096071s1.pdf DISCRIMINATORY XPERT ULTRA DATA3

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Utilization of molecular resistance test results as tools to support public health efforts for improved control of rifampicin-resistant tuberculosis

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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)UvA-DARE (Digital Academic Repository)Utilization of molecular resistance test results as tools to support public healthefforts for improved control of rifampicin-resistant tuberculosisNg, K.C.S.Publication date2020Document VersionOther versionLicenseOtherLink to publicationCitation for published version (APA):Ng, K. C. S. (2020). Utilization of molecular resistance test results as tools to support publichealth efforts for improved control of rifampicin-resistant tuberculosis. [Thesis, fully internal,Universiteit van Amsterdam].General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s)and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an opencontent license (like Creative Commons).Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, pleaselet the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the materialinaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letterto: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. Youwill be contacted as soon as possible.Download date:10 Mar 202363Chapter 3 Xpert Ultra can unambiguously identify specific rifampicin resistance-conferring mutationsKamela C. S. Ng,a Armand van Deun, a,b Conor J. Meehan,a Gabriela Torrea,a Michèle Driesen,a Siemon Gabriëls,a Leen Rigouts,a,c Emmanuel André,d* Bouke C. de Jonga* Unit of Mycobacteriology, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgiuma; International Union Against Tuberculosis and Lung Disease, Paris, France b; Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgiumc; Laboratory of Clinical Bacteriology and Mycology, KU Leuven, Leuven, Belgiumd* joint final authorsJournal of Clinical Microbiology 2018, 56:e00686-18. https://pubmed.ncbi.nlm.nih.gov/2992564364INTRODUCTIONThe deluge of data produced by XpertMTB/RIF (Cepheid) can help improve global rifampicin-resistant tuberculosis (RR-TB) control strategies through molecular epidemiological surveillance (1, 2). Recently, a new version of the test – Xpert Ultra (hereinafter called Ultra) was released (3). Determining the relationship between RR-conferring rpoB mutations, Ultra probes, and melting temperature shifts (∆Tm) – the difference between mutant and wildtype melting temperatures – allows Ultra results to be utilized for rapid detection of RR-TB strains and related underlying rpoB mutations. METHODSTo validate the usefulness of Ultra results for predicting specific mutations, we analyzed 10 RS-TB and 107 RR-TB strains from the Belgian Coordinated Collections of Microorganisms in the Institute of Tropical Medicine, Antwerp, Belgium. These strains harbor 36 unique RR-conferring mutations determined by rpoB sequencing.RESULTSOverall, 31/32 (97%) mutations inside the Rifampicin Resistance Determining Region (RRDR) were correctly identified by Ultra. Of concern, mutation His445Arg gave a “RIF Resistance INDERTERMINATE” result among 3/4 strains tested while it was reported as RR in the initial validation study (3). The silent mutation Thr444Thr was not reported as RR (Figure 1). The RR-conferring mutations on codons 170, 250, 299, 482, and 491 situated outside the RRDR were not detected.The probe reactions observed were largely in agreement with previous results (3) albeit we noted that mutations Met434Val, Met434Thr and those in codon 435 were captured only by probe rpoB2; Ser450Leu and Ser450Trp were captured by both probes rpoB3 and rpoB4a, His445Arg was captured only by probe rpoB3; and Lys446Gln was captured only by probe rpoB4.CHAPTER 365Figure 1. Overview of Xpert Ultra test results. The observed probe reactions for each RRDR mutation were laid over the claimed probe coverage (light gray). Shown in black are probe reactions concordant with manufacturer claims, in blue are probe reactions missed by one probe but captured by another probe, and in red is a probe reaction representing a “RIF Resistance Indeterminate” result from 3 out of 4 strains tested. Results in the hatched pattern were superimposed for greater visibility.All mutations except those in codon 450 were associated with a negative ∆Tm (Figure 2). The combination of ∆Tm values with the capturing probes enabled to differentiate mutations in codons 428, 430, 431, 432, 434, 435, 441, 445, 446, and 452, including disputed mutations (4) (Table 1). Mutation Asp435Tyr was unambiguously distinguished from Asp435Val through probe rpoB2|∆Tm|, while mutations Ser441Gln and Ser441Leu were discriminated from the rest by |∆Tm| values of probes rpoB2 and rpoB3. Mutations His445Asp and His445Tyr were distinguished from disputed mutations His445Leu and His445Asn through probe rpoB3 |∆Tm|. Ser450Leu was distinguished from Ser450Trp by probe rpoB4A |∆Tm| except for one strain with an outlier rpoB4A Tm of 70.9°C in contrast to the other 13 strains with rpoB4A Tm of 73.3-73.8°C. The indeterminate result associated with His445Arg may be caused by its |∆Tm|=1.8°C compared with |∆Tm| typically exceeding 2°C for other mutations. DISCRIMINATORY XPERT ULTRA DATA366Figure 2. Melting temperature shifts (ΔTms) observed upon detection of a rifampin resistance (RR)-conferring rpoB mutation in the RR-determining region (RRDR) by Xpert Ultra. The y axis reflects the melting temperature difference (ΔTm) between mutant and wild-type probe-amplicon hybrids, while the x axis shows the mutations that we tested. The data points on the graph are ΔTm values grouped by their associated Ultra probes (differentiated by color), which correspond to a specific rpoB mutation. x axis labels in brown are disputed mutations.CONCLUSIONSOur findings confirm the ability of Ultra to unambiguously identify a wide range of RRDR mutations. With the unprecedented roll-out of XpertMTB/RIF and associated connectivity solutions, such as DataToCare (Savics, Belgium) and GXAlert (SystemOne, USA) (2), Ultra results may be exploited to rule-out transmission between RR-TB patients in a specific setting, distinguish relapse from reinfection, and resolve discordance between an RR Ultra result and a low-level RS phenotypic result due to a disputed mutation. For such applications, it is key that ∆Tm values are included in the exported results.AcknowledgementThis work was supported by Erasmus Mundus Joint Doctorate Fellowship grant 2016-1346 to KCSN.CHAPTER 367Table 1.  Xpert Ultra raw resultsaaCapturing probes, wild-type melt peak temperature (Tm) ranges and means, mutant Tm ranges, and absolute values of melting temperature shift (ΔTm) ranges associated with specific rpoB mutations in the strains tested and the corresponding nucleotide changes. ND, strains that harbored corresponding mutations outside the RRDR yielded a “RIF Resistance Not Detected” result. *, rifampin resistance-determining region (RRDR) mutation unambiguously identified by unique combinations of Ultra probes and ΔTms, including disputed ones (in italics). #, rifampin susceptible according to phenotypic testing.bFor double mutants, the high-confidence RR-conferring mutations are underlined (6, 7). DISCRIMINATORY XPERT ULTRA DATA368REFERENCES1. Andre E, Isaacs C, Affolabi D, Alagna R, Brockmann D, de Jong BC, Cambau E, Churchyard G, Cohen T, Delmee M, Delvenne JC, Farhat M, Habib A, Holme P, Keshavjee S, Khan A, Lightfoot P, Moore D, Moreno Y, Mundade Y, Pai M, Patel S, Nyaruhirira AU, Rocha LE, Takle J, Trebucq A, Creswell J, Boehme C. 2016. Connectivity of diagnostic technologies: improving surveillance and accelerating tuberculosis elimination. Int J Tuberc Lung Dis 20:999-1003.2. Ng KC, Meehan CJ, Torrea G, Goeminne L, Diels M, Rigouts L, de Jong BC, Andre E. 2018. Potential Application of Digitally Linked Tuberculosis Diagnostics for Real-Time Surveillance of Drug-Resistant Tuberculosis Transmission: Validation and Analysis of Test Results. JMIR Med Inform 6:e12.3. Chakravorty S, Simmons AM, Rowneki M, Parmar H, Cao Y, Ryan J, Banada PP, Deshpande S, Shenai S, Gall A, Glass J, Krieswirth B, Schumacher SG, Nabeta P, Tukvadze N, Rodrigues C, Skrahina A, Tagliani E, Cirillo DM, Davidow A, Denkinger CM, Persing D, Kwiatkowski R, Jones M, Alland D. 2017. The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing. MBio 8.4. Van Deun A, Aung KJ, Bola V, Lebeke R, Hossain MA, de Rijk WB, Rigouts L, Gumusboga A, Torrea G, de Jong BC. 2013. Rifampin drug resistance tests for tuberculosis: challenging the gold standard. J Clin Microbiol 51:2633-40.CHAPTER 369SUPPLEMENTARY INFORMATIONSupplemental methods. Processing of rifampicin-resistant tuberculosis thermolysates for Xpert Ultratesting: methodological details.Figure S1. Interpreting Xpert Ultra ΔTm plus probe reactions observed in 2 rifampin-resistant tuberculosis patients for evaluation of a potential transmission event.FigureS2. Interpreting Xpert Ultra ΔTm plus probe reactions observed in 2 rifampin-resistant tuberculosis patients for identification of relapse cases.https://jcm.asm.org/highwire/filestream/123403/field_highwire_adjunct_files/0/zjm999096071s1.pdf DISCRIMINATORY XPERT ULTRA DATA3

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Utilization of molecular resistance test results as tools to support public health efforts for improved control of rifampicin-resistant tuberculosis

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