Mortality from drug-resistant tuberculosis in high-burden countries comparing routine drug susceptibility testing with whole-genome sequencing: a multicentre cohort study.

BACKGROUND: Drug resistance threatens global tuberculosis control. We aimed to examine mortality in patients with tuberculosis from high-burden countries, according to concordance or discordance of results from drug susceptibility testing done locally and whole-genome sequencing (WGS). METHODS: In this multicentre cohort study, we collected pulmonary Mycobacterium tuberculosis isolates and clinical data from individuals with tuberculosis from antiretroviral therapy programmes and tuberculosis clinics in Côte d'Ivoire, Democratic Republic of the Congo, Kenya, Nigeria, Peru, South Africa, and Thailand, stratified by HIV status and drug resistance. Sites tested drug susceptibility using routinely available methods. WGS was done on Illumina HiSeq 2500 in the USA and Switzerland, and TBprofiler was used to analyse the genomes. We included individuals aged 16 years or older with pulmonary tuberculosis (bacteriologically confirmed or clinically diagnosed). We analysed mortality in multivariable logistic regression models adjusted for sex, age, HIV status, history of tuberculosis, and sputum positivity. FINDINGS: Between Sept 1, 2014, and July 4, 2016, of 634 patients included in our previous analysis, we included 582 patients with tuberculosis (median age 33 years [IQR 27-43], 225 [39%] women, and 247 [42%] HIV-positive). Based on WGS, 339 (58%) isolates were pan-susceptible, 35 (6%) monoresistant, 146 (25%) multidrug-resistant, and 24 (4%) pre-extensively drug-resistant (pre-XDR) or XDR. The analysis of mortality was based on 530 patients; 63 (12%) died and 77 (15%) patients received inappropriate treatment. Mortality ranged from 6% (18 of 310) in patients with pan-susceptible tuberculosis to 39% (nine of 23) in patients with pre-XDR or XDR tuberculosis. The adjusted odds ratio for mortality was 4·92 (95% CI 2·47-9·78) among undertreated patients, compared with appropriately treated patients. INTERPRETATION: In seven countries with a high burden of tuberculosis, we observed discrepancies between drug resistance patterns obtained locally and WGS. The underdiagnosis of drug resistance resulted in inappropriate treatment and higher mortality. WGS can provide accurate and detailed drug resistance information required to improve the outcomes of drug-resistant tuberculosis in high-burden settings. Our results support WHO's call for point-of-care tests based on WGS. FUNDING: National Institutes of Allergy and Infectious Diseases, Swiss National Science Foundation, and Swiss National Center for Mycobacteria.

Natural Polymorphisms in Mycobacterium tuberculosis Conferring Resistance to Delamanid in Drug-Naive Patients.

Mutations in the genes of the F420 signaling pathway of Mycobacterium tuberculosis complex, including dnn, fgd1, fbiA, fbiB, fbiC, and fbiD, can lead to delamanid resistance. We searched for such mutations among 129 M. tuberculosis strains from Asia, South America, and Africa using whole-genome sequencing; 70 (54%) strains had at least one mutation in one of the genes. For 10 strains with mutations, we determined the MIC of delamanid. We found one strain from a delamanid-naive patient carrying the natural polymorphism Tyr29del (ddn) that was associated with a critical delamanid MIC.

Drug susceptibility testing and mortality in patients treated for tuberculosis in high-burden countries: a multicentre cohort study.

BACKGROUND: Drug resistance is a challenge for the global control of tuberculosis. We examined mortality in patients with tuberculosis from high-burden countries, according to concordance or discordance of results from drug susceptibility testing done locally and in a reference laboratory. METHODS: This multicentre cohort study was done in Côte d'Ivoire, Democratic Republic of the Congo, Kenya, Nigeria, South Africa, Peru, and Thailand. We collected Mycobacterium tuberculosis isolates and clinical data from adult patients aged 16 years or older. Patients were stratified by HIV status and tuberculosis drug resistance. Molecular or phenotypic drug susceptibility testing was done locally and at the Swiss National Center for Mycobacteria, Zurich, Switzerland. We examined mortality during treatment according to drug susceptibility test results and treatment adequacy in multivariable logistic regression models adjusting for sex, age, sputum microscopy, and HIV status. FINDINGS: We obtained M tuberculosis isolates from 871 patients diagnosed between 2013 and 2016. After exclusion of 237 patients, 634 patients with tuberculosis were included in this analysis; the median age was 33·2 years (IQR 26·9-42·5), 239 (38%) were women, 272 (43%) were HIV-positive, and 69 (11%) patients died. Based on the reference laboratory drug susceptibility test, 394 (62%) strains were pan-susceptible, 45 (7%) monoresistant, 163 (26%) multidrug-resistant (MDR), and 30 (5%) had pre-extensively or extensively drug resistant (pre-XDR or XDR) tuberculosis. Results of reference and local laboratories were concordant for 513 (81%) of 634 patients and discordant for 121 (19%) of 634. Overall, sensitivity to detect any resistance was 90·8% (95% CI 86·5-94·2) and specificity 84·3% (80·3-87·7). Mortality ranged from 6% (20 of 336) in patients with pan-susceptible tuberculosis treated according to WHO guidelines to 57% (eight of 14) in patients with resistant strains who were under-treated. In logistic regression models, compared with concordant drug susceptibility test results, the adjusted odds ratio of death was 7·33 (95% CI 2·70-19·95) for patients with discordant results potentially leading to under-treatment. INTERPRETATION: Inaccurate drug susceptibility testing by comparison with a reference standard leads to under-treatment of drug-resistant tuberculosis and increased mortality. Rapid molecular drug susceptibility test of first-line and second-line drugs at diagnosis is required to improve outcomes in patients with MDR tuberculosis and pre-XDR or XDR tuberculosis. FUNDING: National Institutes of Allergy and Infectious Diseases, Swiss National Science Foundation, Swiss National Center for Mycobacteria.

Feasibility of the TBDx automated digital microscopy system for the diagnosis of pulmonary tuberculosis.

BACKGROUND: Improved and affordable diagnostic or triage tests are urgently needed at the microscopy centre level. Automated digital microscopy has the potential to overcome issues related to conventional microscopy, including training time requirement and inconsistencies in results interpretation. METHODS: For this blinded prospective study, sputum samples were collected from adults with presumptive pulmonary tuberculosis in Lima, Peru and Ho Chi Minh City, Vietnam. TBDx performance was evaluated as a stand-alone and as a triage test against conventional microscopy and Xpert, with culture as the reference standard. Xpert was used to confirm positive cases. FINDINGS: A total of 613 subjects were enrolled between October 2014 and March 2015, with 539 included in the final analysis. The sensitivity of TBDx was 62·2% (95% CI 56·6-67·4) and specificity was 90·7% (95% CI 85·9-94·2) compared to culture. The algorithm assessing TBDx as a triage test achieved a specificity of 100% while maintaining sensitivity. INTERPRETATION: While the diagnostic performance of TBDx did not reach the levels obtained by experienced microscopists in reference laboratories, it is conceivable that it would exceed the performance of less experienced microscopists. In the absence of highly sensitive and specific molecular tests at the microscopy centre level, TBDx in a triage-testing algorithm would optimize specificity and limit overall cost without compromising the number of patients receiving up-front drug susceptibility testing for rifampicin. However, the algorithm would miss over one third of patients compared to Xpert alone.

FIND Tuberculosis Strain Bank: a Resource for Researchers and Developers Working on Tests To Detect Mycobacterium tuberculosis and Related Drug Resistance.

The spread of multidrug-resistant (MDR) tuberculosis (TB) and extensively drug-resistant (XDR) TB hampers global efforts in the fight against tuberculosis. To enhance the development and evaluation of diagnostic tests quickly and efficiently, well-characterized strains and samples from drug-resistant tuberculosis patients are necessary. In this project, the Foundation for Innovative New Diagnostics (FIND) has focused on the collection, characterization, and storage of such well-characterized reference materials and making them available to researchers and developers. The collection is being conducted at multiple centers in Southeast Asia, South America, Eastern Europe, and soon the sub-Saharan Africa regions. Strains are characterized for their phenotypic resistances and MICs to first-line drugs (FLDs) and second-line drugs (SLDs) using the automated MGIT 960 system following validated procedures and WHO criteria. Analysis of resistance-associated mutations is done by whole-genome sequencing (WGS) using the Illumina NextSeq system. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis and WGS are used to determine strain lineages. All strains are maintained frozen at -80°C ± 10°C as distinct mother and daughter lots. All strains are extensively quality assured. The data presented here represent an analysis of the initial part of the collection. Currently, the bank contains 118 unique strains with extracted genomic DNA and matched sputum, serum, and plasma samples and will be expanded to a minimum of 1,000 unique strains over the next 3 years. Analysis of the current strains by phenotypic resistance testing shows 102 (86.4%), 10 (8.5%), and 6 (5.1%) MDR, XDR, and mono/poly resistant strains, respectively. Two of the strains are resistant to all 11 drugs that were phenotypically tested. WGS mutation analysis revealed FLD resistance-associated mutations in the rpoB, katG, inhA, embB, embA, and pncA genes; SLD resistance in the gyrA, gyrB, rrs, eis, and tlyA genes; and ethionamide resistance in the ethA genes. Most important lineages are represented in the bank, and further collections have been initiated to increase geographic and lineage diversity. The bank provides highly characterized and high-quality strains as a resource for researchers and developers in support of the development and evaluation of new diagnostics and drug resistance detection tools.

Rapid Detection of Mycobacterium tuberculosis Strains Resistant to Isoniazid and/or Rifampicin: Standardization of Multiplex Polymerase Chain Reaction Analysis.

Drug susceptibility testing using molecular techniques can enhance the identification of drug-resistant Mycobacterium tuberculosis Two multiplex real-time polymerase chain reaction (qPCR) assays were developed to detect the most common resistance-associated mutations to isoniazid (katGS315T, inhA-15C → T), and rifampicin (rpoBH526Y and rpoBS531L). To assess the species specificity of the qPCR, we selected 31 nontuberculous mycobacteria (NTM) reference strains belonging to 17 species from the public collection of mycobacterial cultures (BCCM/ITM). Additionally, we tested 17 isoniazid and/or rifampicin-resistant strains with other mutations in the target genes to assess mutation specificity. The limit of detection for all the targeted mutations was 20 bacilli/reaction. Multiplex 1 showed 90%, 95%, and 100% efficiency for wild type (WT), Mut katGS315T, and Mut rpoBS531L, respectively; whereas Multiplex 2 showed 97%, 94%, and 90% efficiency for WT, Mut inhA-15, and Mut rpoBH526Y, respectively. Three of 17 strains that presented other mutations in the target genes were identified as rifampicin resistant and only 3/31 NTM showed a similar melting temperature to rpoBL531 and/or katGT315 mutants. Thus, our proposed cascade of specific tuberculosis detection followed by drug resistance testing showed sensitivities for katGS315T, rpoBS531L, rpoBH526Y, and inhA-15 detection of 100%, 100%, 100%, and 96%, respectively; and specificities of 98%, 95%, 100%, and 100, respectively.

Standardization of a TaqMan-based real-time PCR for the detection of Mycobacterium tuberculosis-complex in human sputum.

Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Löwenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P < 0.05). The qPCR sensitivity was similar (P = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost ∼5US$ per sample and provided same-day results compared with 2-6 weeks for culture.

Genetic variability of Mycobacterium tuberculosis complex in patients with no known risk factors for MDR-TB in the North-Eastern part of Lima, Peru.

BACKGROUND: The aim of this study was to investigate the genetic diversity among Mycobacterium tuberculosis complex circulating in patients with no known risk factors for multi-drug resistant (MDR) tuberculosis (TB) living in a high MDR burden area and analyze the relationship between genotypes, primary drug resistance and age. METHODS: Samples were collected during January-July 2009. Isolates were tested for drug susceptibility to first-line drugs and were genotyped by spoligotyping and the 15-loci Mycobacterial Interspersed Repetitive Unit (MIRU15). RESULTS: Among the 199 isolates analyzed, 169 (84.9%) were identified in the SpolDB4.0 and 30 (15.1%) could not be matched to any lineage. The most prevalent lineage was Haarlem (29.6%), followed by T (15.6%), Beijing (14.1%), Latin American Mediterranean (12.6%) and U (8.5%). A few isolates belonged to the X and S clades (4.5%). Spoligotype analysis identified clustering among 148 of 169 isolates, whereas with MIRU15 all isolates were unique. Out of 197 strains; 31.5% were resistant to at least one drug, 7.5% were MDR and 22.3% showed any resistance to isoniazid. CONCLUSION: In contrast with other Latin-American countries where LAM lineage is the most predominant, we found the spoligotype 50 from the Haarlem lineage as the most common. None of the prevailing lineages showed a significant association with age or resistance to isoniazid and/or rifampicin.