A Nanopore Sequencing-based Pharmacogenomic Panel to Personalize Tuberculosis Drug Dosing.

Rationale: Standardized dosing of antitubercular drugs leads to variable plasma drug levels, which are associated with adverse drug reactions, delayed treatment response, and relapse. Mutations in genes affecting drug metabolism explain considerable interindividual pharmacokinetic variability; however, pharmacogenomic assays that predict metabolism of antitubercular drugs have been lacking. Objectives: We sought to develop a Nanopore sequencing panel and validate its performance in patients with active tuberculosis (TB) to personalize treatment dosing. Methods: We developed a Nanopore sequencing panel targeting 15 SNPs in five genes affecting the metabolism of antitubercular drugs. For validation, we sequenced DNA samples (n = 48) from the 1,000 Genomes Project and compared the variant calling accuracy with that of Illumina genome sequencing. We then sequenced DNA samples from patients with active TB (n = 100) from South Africa on a MinION Mk1C and evaluated the relationship between genotypes and pharmacokinetic parameters for isoniazid (INH) and rifampin (RIF). Measurements and Main Results: The pharmacogenomic panel achieved 100% concordance with Illumina sequencing in variant identification for the samples from the 1,000 Genomes Project. In the clinical cohort, coverage was more than 100× for 1,498 of 1,500 (99.8%) amplicons across the 100 samples. Thirty-three percent, 47%, and 20% of participants were identified as slow, intermediate, and rapid INH acetylators, respectively. INH clearance was 2.2 times higher among intermediate acetylators and 3.8 times higher among rapid acetylators, compared with slow acetylators (P < 0.0001). RIF clearance was 17.3% (2.50-29.9) lower in individuals with homozygous AADAC rs1803155 G→A substitutions (P = 0.0015). Conclusions: Targeted sequencing can enable the detection of polymorphisms that influence TB drug metabolism on a low-cost, portable instrument to personalize dosing for TB treatment or prevention.

Multi-level tuberculosis of the spine identified by 18 F-FDG-PET/CT and concomitant urogenital tuberculosis: a case report from the spinal TB X cohort.

BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and typically infects the lungs. However, extrapulmonary forms of TB can be found in approximately 20% of cases. It is suggested, that up to 10% of extrapulmonary TB affects the musculoskeletal system, in which spinal elements (spinal tuberculosis, STB) are involved in approximately 50% of the cases. STB is a debilitating disease with nonspecific symptoms and diagnosis is often delayed for months to years. In our Spinal TB X Cohort, we aim to describe the clinical phenotype of STB using whole-body 18 F-fluorodeoxyglucose positron emission tomography computed tomography (PET/CT) and to identify a specific gene expression profile for the different stages of dissemination on PET/CT. Here we report on the first patient recruited into our cohort who underwent PET/CT before treatment initiation, at 6-months and at 12-months - time of TB treatment completion. CASE PRESENTATION: A 27-year-old immunocompetent male presented with severe thoracolumbar back pain for 9 months with severe antalgic gait and night sweats. Magnetic resonance imaging (MRI) of the whole spine revealed multilevel spinal disease (T5/6, T11/12, L3/4) in keeping with STB. After informed consent and recruitment into the Spinal TB X Cohort, the patient underwent PET/CT as per protocol, which revealed isolated multilevel STB (T4-7, T11/12, L3/4) with no concomitant lung or urogenital lesion. However, sputum and urine were Xpert MTB/RIF Ultra positive and Mtb was cultured from the urine sample. CT-guided biopsy of the T11/12 lesion confirmed drug-sensitive Mtb on Xpert MTB/RIF Ultra and the patient was started on TB treatment according to local guidelines for 12 months. The 6-month follow-up PET/CT revealed new and existing spinal lesions with increased FDG-uptake despite significant improvement of clinical features and laboratory markers. After 9 months of treatment, the patient developed an acute urethral stricture, most likely due to urogenital TB, and a suprapubic catheter was inserted. The 12-month PET/CT showed significantly decreased PET/CT values of all lesions, however, significant persistent spinal inflammation was present at the end of TB treatment. Clinically, the patient was considered cured by the TB control program and currently awaits urethroplasty. CONCLUSIONS: In our case, PET/CT emerged as a valuable imaging modality for the initial assessment, surpassing MRI by revealing more comprehensive extensive disease. Subsequent PET/CT scans at 6-month uncovered new lesions and increased inflammation in existing ones, while by the end of TB treatment, all lesions exhibited improvement. However, the interpretation of FDG avidity remains ambiguous, whether it correlates with active infection and viable Mtb. or fibro- and osteoblast activity indicative of the healing process. Additionally, the absence of extraspinal TB lesions on PET/CT despite positive microbiology from sputum and urine maybe explained by paucibacillary, subclinical infection of extraspinal organs. The Spinal TB X Cohort endeavours to shed light on whole-body imaging patterns at diagnosis, their evolution midway through TB treatment, and upon treatment completion. Ultimately, this study aims to advance our understanding of the biology of this complex disease.

Evaluation of a Rapid Molecular Drug-Susceptibility Test for Tuberculosis.

BACKGROUND: Fluoroquinolones and second-line injectable drugs are the backbone of treatment regimens for multidrug-resistant tuberculosis, and resistance to these drugs defines extensively drug-resistant tuberculosis. We assessed the accuracy of an automated, cartridge-based molecular assay for the detection, directly from sputum specimens, of Mycobacterium tuberculosis with resistance to fluoroquinolones, aminoglycosides, and isoniazid. METHODS: We conducted a prospective diagnostic accuracy study to compare the investigational assay against phenotypic drug-susceptibility testing and DNA sequencing among adults in China and South Korea who had symptoms of tuberculosis. The Xpert MTB/RIF assay and sputum culture were performed. M. tuberculosis isolates underwent phenotypic drug-susceptibility testing and DNA sequencing of the genes katG, gyrA, gyrB, and rrs and of the eis and inhA promoter regions. RESULTS: Among the 308 participants who were culture-positive for M. tuberculosis, when phenotypic drug-susceptibility testing was used as the reference standard, the sensitivities of the investigational assay for detecting resistance were 83.3% for isoniazid (95% confidence interval [CI], 77.1 to 88.5), 88.4% for ofloxacin (95% CI, 80.2 to 94.1), 87.6% for moxifloxacin at a critical concentration of 0.5 µg per milliliter (95% CI, 79.0 to 93.7), 96.2% for moxifloxacin at a critical concentration of 2.0 µg per milliliter (95% CI, 87.0 to 99.5), 71.4% for kanamycin (95% CI, 56.7 to 83.4), and 70.7% for amikacin (95% CI, 54.5 to 83.9). The specificity of the assay for the detection of phenotypic resistance was 94.3% or greater for all drugs except moxifloxacin at a critical concentration of 2.0 µg per milliliter (specificity, 84.0% [95% CI, 78.9 to 88.3]). When DNA sequencing was used as the reference standard, the sensitivities of the investigational assay for detecting mutations associated with resistance were 98.1% for isoniazid (95% CI, 94.4 to 99.6), 95.8% for fluoroquinolones (95% CI, 89.6 to 98.8), 92.7% for kanamycin (95% CI, 80.1 to 98.5), and 96.8% for amikacin (95% CI, 83.3 to 99.9), and the specificity for all drugs was 99.6% (95% CI, 97.9 to 100) or greater. CONCLUSIONS: This investigational assay accurately detected M. tuberculosis mutations associated with resistance to isoniazid, fluoroquinolones, and aminoglycosides and holds promise as a rapid point-of-care test to guide therapeutic decisions for patients with tuberculosis. (Funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the Ministry of Science and Technology of China; ClinicalTrials.gov number, NCT02251327 .).

Linezolid resistance in patients with drug-resistant TB and treatment failure in South Africa.

OBJECTIVES: Limited data exist on clinical associations and genotypic correlates of linezolid resistance in Mycobacterium tuberculosis. We aimed to describe mutations and clinical factors associated with phenotypic linezolid resistance from patients with drug-resistant TB at two public sector facilities in South Africa. METHODS: Adults and adolescents with treatment failure (culture positivity ≥4 months) on a linezolid-containing regimen were retrospectively identified. Phenotypic resistance, as defined by a linezolid MIC >1 mg/L, was assessed for retrieved isolates using broth microdilution. Targeted sequencing of rrl and rplC was performed, irrespective of growth on subculture. RESULTS: Thirty-nine patients with linezolid-based treatment failure were identified, 13 (33%) of whom had phenotypic or genotypic linezolid resistance after a median duration of 22 months (range = 7-32) of linezolid therapy. Paired MIC testing and genotyping was performed on 55 unique isolates. All isolates with phenotypic resistance (n = 16) were associated with known resistance mutations, most frequently due to the T460C substitution in rplC (n = 10); rrl mutations included G2814T, G2270C/T and A2810C. No mutations were detected in isolates with MICs at or below the critical concentration. CONCLUSIONS: Linezolid resistance occurred in a third of patients with drug-resistant TB and treatment failure. Resistance occurred late and was predicted by a limited number of mutations in rrl and rplC. Screening for genotypic resistance should be considered for patients with a positive culture after 4 months of linezolid therapy in order to optimize treatment and avoid the toxicity of ineffective linezolid therapy.

Fitness costs of rifampicin resistance in Mycobacterium tuberculosis are amplified under conditions of nutrient starvation and compensated by mutation in the ß' subunit of RNA polymerase.

Rifampicin resistance, a defining attribute of multidrug-resistant tuberculosis, is conferred by mutations in the ß subunit of RNA polymerase. Sequencing of rifampicin-resistant (RIF-R) clinical isolates of Mycobacterium tuberculosis revealed, in addition to RIF-R mutations, enrichment of potential compensatory mutations around the double-psi ß-barrel domain of the ß' subunit comprising the catalytic site and the exit tunnel for newly synthesized RNA. Sequential introduction of the resistance allele followed by the compensatory allele in isogenic Mycobacterium smegmatis showed that these mutations respectively caused and compensated a starvation enhanced growth defect by altering RNA polymerase activity. While specific combinations of resistance and compensatory alleles converged in divergent lineages, other combinations recurred among related isolates suggesting transmission of compensated RIF-R strains. These findings suggest nutrient poor growth conditions impose larger selective pressure on RIF-R organisms that results in the selection of compensatory mutations in a domain involved in catalysis and starvation control of RNA polymerase transcription.

Linezolid for treatment of chronic extensively drug-resistant tuberculosis.

BACKGROUND: Linezolid has antimycobacterial activity in vitro and is increasingly used for patients with highly drug-resistant tuberculosis. METHODS: We enrolled 41 patients who had sputum-culture-positive extensively drug-resistant (XDR) tuberculosis and who had not had a response to any available chemotherapeutic option during the previous 6 months. Patients were randomly assigned to linezolid therapy that started immediately or after 2 months, at a dose of 600 mg per day, without a change in their background regimen. The primary end point was the time to sputum-culture conversion on solid medium, with data censored 4 months after study entry. After confirmed sputum-smear conversion or 4 months (whichever came first), patients underwent a second randomization to continued linezolid therapy at a dose of 600 mg per day or 300 mg per day for at least an additional 18 months, with careful toxicity monitoring. RESULTS: By 4 months, 15 of the 19 patients (79%) in the immediate-start group and 7 of the 20 (35%) in the delayed-start group had culture conversion (P=0.001). Most patients (34 of 39 [87%]) had a negative sputum culture within 6 months after linezolid had been added to their drug regimen. Of the 38 patients with exposure to linezolid, 31 (82%) had clinically significant adverse events that were possibly or probably related to linezolid, including 3 patients who discontinued therapy. Patients who received 300 mg per day after the second randomization had fewer adverse events than those who continued taking 600 mg per day. Thirteen patients completed therapy and have not had a relapse. Four cases of acquired resistance to linezolid have been observed. CONCLUSIONS: Linezolid is effective at achieving culture conversion among patients with treatment-refractory XDR pulmonary tuberculosis, but patients must be monitored carefully for adverse events. (Funded by the National Institute of Allergy and Infectious Diseases and the Ministry of Health and Welfare, South Korea; ClinicalTrials.gov number, NCT00727844.).

Predictors of pulmonary tuberculosis treatment outcomes in South Korea: a prospective cohort study, 2005-2012.

BACKGROUND: Tuberculosis remains an important health concern in many countries. The aim of this study was to identify predictors of unfavorable outcomes at the end of treatment (EOT) and at the end of study (EOS; 40 months after EOT) in South Korea. METHODS: New or previously treated tuberculosis patients were recruited into a prospective observational cohort study at two hospitals in South Korea. To identify predictors of unfavorable outcomes at EOT and EOS, logistic regression analysis was performed. RESULTS: The proportion of multidrug-resistant tuberculosis (MDR-TB) was 8.2% in new cases and 57.9% in previously treated cases. Of new cases, 68.6% were cured, as were 40.7% of previously treated cases. At EOT, diabetes, ≥3 previous TB episodes, ≥1 significant regimen change, and MDR-TB were significantly associated with treatment failure or death. At EOS, age ≥35, body-mass index (BMI) <18.5, diabetes, and MDR-TB were significantly associated with treatment failure, death, or relapse. Among cases that were cured at EOT, age ≥50 and a BMI <18.5 were associated with subsequent death or relapse during follow-up to EOS. Treatment interruption was associated with service sector employees or laborers, bilateral lesions on chest X-ray, and previous treatment failure or treatment interruption history. CONCLUSIONS: Risk factors for poor treatment outcomes at EOT and EOS include both patient factors (diabetes status, age, BMI) and disease factors (history of multiple previous treatment episodes, MDR-TB). In this longitudinal, observational cohort study, diabetes mellitus and MDR-TB were risk factors for poor treatment outcomes and relapse. Measures to help ensure that the first tuberculosis treatment episode is also the last one may improve treatment outcomes. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT00341601.

Comparing gene expression profiles of adults with isolated spinal tuberculosis to disseminated spinal tuberculosis identified by 18FDG-PET/CT at time of diagnosis, 6- and 12-months follow-up: classifying clinical stages of spinal tuberculosis and monitoring treatment response (Spinal TB X cohort study).

BACKGROUND: Tuberculosis (TB) is one of the top ten causes of death worldwide, with approximately 10 million cases annually. Focus has been on pulmonary TB, while extrapulmonary TB (EPTB) has received little attention. Diagnosis of EPTB remains challenging due to the invasive procedures required for sample collection. Spinal TB (STB) accounts for 10% of EPTB and often leads to lifelong debilitating disease due to devastating spinal deformation and compression of neural structures. Little is known about the extent of disease, although both isolated STB and a disseminated form of STB have been described. In our Spinal TB X cohort study, we aim to describe the clinical phenotype of STB using whole-body 18FDG-PET/CT, identify a specific gene expression profile for different stages of dissemination and compare findings to previously described gene expression signatures for latent and active pulmonary TB. METHODS: A single-centre, prospective cohort study will be established to describe the distributional pattern of STB detected by whole-body 18FDG-PET/CT and gene expression profile of patients with suspected STB on magnetic resonance imaging (MRI) at point of diagnosis, six months, and 12 months. Blood biobanking will be performed at these time points. Specimens for microbiology will be obtained from sputum/urine, from easily accessible sites of disease (e.g., lymph nodes, abscess) identified in the first 18FDG-PET/CT, from CT-guided biopsy and/or surgery. Clinical parameters and functional scores will be collected at every physical visit. Data will be entered into RedCap® database; data cleaning, validation and analysis will be performed by the study team. The University of Cape Town Ethics Committee approved the protocol (243/2022). DISCUSSION: The Spinal TB X cohort study is the first prospective cohort study using whole-body 18FDG-PET/CT scans in patients with microbiologically confirmed spinal tuberculosis. Dual imaging techniques of the spine using 18FDG-PET/CT and magnetic resonance imaging as well as tissue diagnosis (microbiology and histopathology) will allow us to develop a virtual biopsy model. If successful, a distinct gene-expression profile will aid in blood-based diagnosis (point of care testing) as well as treatment monitoring and would lead to earlier diagnosis of this devastating disease. TRIAL REGISTRATION: The study has been registered on ClinicalTrials.gov (NCT05610098).

Exploring alternative biomaterials for diagnosis of pulmonary tuberculosis in HIV-negative patients by use of the GeneXpert MTB/RIF assay.

The utility of the GeneXpert MTB/RIF (Xpert) assay for detection of Mycobacterium tuberculosis in sputum samples has been extensively studied. However, the performance of the Xpert assay as applied to other readily accessible body fluids such as exhaled breath condensate (EBC), saliva, urine, and blood has not been established. We used the Xpert assay to test EBC, saliva, urine, and blood samples from HIV-negative, smear- and culture-positive pulmonary tuberculosis (TB) patients for the presence of M. tuberculosis. To compare the ability of the assay to perform bacterial load measurements on sputum samples with versus without sample processing, the assay was also performed on paired direct and processed sputum samples from each patient. The Xpert assay detected M. tuberculosis in none of the 26 EBC samples (sensitivity, 0.0%; 95% confidence interval [95% CI], 0.0%, 12.9%), 10 of the 26 saliva samples (sensitivity, 38.5%; 95% CI, 22.4%, 57.5%), 1 of 26 urine samples (sensitivity, 3.8%; 95% CI, 0.7%, 18.9%), and 2 of 24 blood samples (sensitivity, 8.3%; 95% CI, 2.3%, 25.8%). For bacterial load measurements in the different types of sputum samples, the cycle thresholds of the two M. tuberculosis-positive sputum types were well correlated (Spearman correlation of 0.834). This study demonstrates that the Xpert assay should not be routinely used to detect M. tuberculosis in EBC, saliva, urine, or blood samples from HIV-negative patients suspected of having pulmonary tuberculosis. As a test of bacterial load, the assay produced similar results when used to test direct versus processed sputum samples. Sputum remains the optimal sample type for diagnosing pulmonary tuberculosis in HIV-negative patients with the Xpert assay.