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Evaluating culture-free targeted next-generation sequencing for diagnosing drug-resistant tuberculosis: a multicentre clinical study of two end-to-end commercial workflows.

BACKGROUND: Drug-resistant tuberculosis remains a major obstacle in ending the global tuberculosis epidemic. Deployment of molecular tools for comprehensive drug resistance profiling is imperative for successful detection and characterisation of tuberculosis drug resistance. We aimed to assess the diagnostic accuracy of a new class of molecular diagnostics for drug-resistant tuberculosis. METHODS: We conducted a prospective, cross-sectional, multicentre clinical evaluation of the performance of two targeted next-generation sequencing (tNGS) assays for drug-resistant tuberculosis at reference laboratories in three countries (Georgia, India, and South Africa) to assess diagnostic accuracy and index test failure rates. Eligible participants were aged 18 years or older, with molecularly confirmed pulmonary tuberculosis, and at risk for rifampicin-resistant tuberculosis. Sensitivity and specificity for both tNGS index tests (GenoScreen Deeplex Myc-TB and Oxford Nanopore Technologies [ONT] Tuberculosis Drug Resistance Test) were calculated for rifampicin, isoniazid, fluoroquinolones (moxifloxacin, levofloxacin), second line-injectables (amikacin, kanamycin, capreomycin), pyrazinamide, bedaquiline, linezolid, clofazimine, ethambutol, and streptomycin against a composite reference standard of phenotypic drug susceptibility testing and whole-genome sequencing. FINDINGS: Between April 1, 2021, and June 30, 2022, 832 individuals were invited to participate in the study, of whom 720 were included in the final analysis (212, 376, and 132 participants in Georgia, India, and South Africa, respectively). Of 720 clinical sediment samples evaluated, 658 (91%) and 684 (95%) produced complete or partial results on the GenoScreen and ONT tNGS workflows, respectively, with 593 (96%) and 603 (98%) of 616 smear-positive samples producing tNGS sequence data. Both workflows had sensitivities and specificities of more than 95% for rifampicin and isoniazid, and high accuracy for fluoroquinolones (sensitivity approximately ≥94%) and second line-injectables (sensitivity 80%) compared with the composite reference standard. Importantly, these assays also detected mutations associated with resistance to critical new and repurposed drugs (bedaquiline, linezolid) not currently detectable by any other WHO-recommended rapid diagnostics on the market. We note that the current format of assays have low sensitivity (≤50%) for linezolid and more work on mutations associated with drug resistance is needed. INTERPRETATION: This multicentre evaluation demonstrates that culture-free tNGS can provide accurate sequencing results for detection and characterisation of drug resistance from Mycobacterium tuberculosis clinical sediment samples for timely, comprehensive profiling of drug-resistant tuberculosis. FUNDING: Unitaid.

Humans↗

Arthritis associated with tuberculosis.

There has been a resurgence in tuberculosis (TB) worldwide. Approximately 2 billion people have latent infection, 8 million would develop active TB annually, and 2-3 million would die due to TB. With this resurgence, cases with extrapulmonary TB have also shown an increase. Approximately 10-11% of extrapulmonary TB involves joints and bones, which is approximately 1-3% of all TB cases. The global prevalence of latent joint and bone TB is approximately 19-38 million.TB arthritis most commonly manifests as a monoarthritis of weight-bearing joints in the hip or the knee. Oligo- or polyarticular presentation is not rare and may cause diagnostic confusion with inflammatory arthritis. Owing to the low incidence in developed countries, the diagnosis of joint and bone TB is often delayed. A high degree of sensitivity to this diagnosis would prevent delays, permitting prompt institution of anti-TB therapy and preventing irreversible joint damage. Despite advances, confirmation of diagnosis still relies on lengthy microbiological techniques or invasive biopsy. Due to the frequency of isoniazid resistance, initial treatment at present typically includes a combination of four drugs: isoniazid, rifampicin, pyrazinamide and streptomycin or ethambutol. Antimicrobial therapy should be of at least 9 months duration, longer in children and immunocompromised hosts. Surgical procedures should be restricted to joints with severe cartilage destruction, large abscesses, joint deformity, multiple drug resistance or atypical mycobacteria.

Antitubercular Agents↗

Antimycobacterial activity of substituted isosteres of pyridine- and pyrazinecarboxylic acids. 2.

Pyridines and pyrazines substituted with 1,2,4-oxadiazole-5-ones, 1,2,4-oxadiazole-5-thiones, and 1,3,4-oxathiazoline-2-ones were synthesized and tested against Mycobacterium tuberculosis. The two former ring systems were documented in the literature to act as carboxylic acid isosteres. The latter series was synthesized as possible synthetic intermediates to 1,2,4-thiadiazole-3-ones and was included in this study due to their interesting activity. Pivaloyloxymethyl derivatives of the isosteres were also prepared in order to increase their lipophilicity and therefore improve their cellular permeability. The derivatized isosteres were expected to be biotransformed by esterases to the active species after penetration of the mycobacterial cell wall. Biological properties of the compounds were compared with the unmodified polar isosteres of pyrazinoic and nicotinic acids. The majority of the compounds exhibited activities ranging from 0.5 to 16 times the potency of pyrazinamide.

Antitubercular Agents↗

Mycobacterium tuberculosis infection and laboratory diagnosis in solid-organ transplant recipients.

Tuberculosis (TB) is an unusual infection in transplant recipients. We evaluated (i) the frequency of TB, (ii) the duration to develop the TB infection, and (iii) clinical consequences, in 380 solid-organ recipients from January 1995 to December 2000. A total of 10 (2.63%) patients (eight renal, two liver transplant recipients) were found to have post-transplantation TB. The frequency of TB in this patient population is 8.5-fold higher than the prevalance in the general Turkish population. Tuberculosis developed within 2-33 months after transplantation, with a median of 15 months. In all of these 10 patients, Mycobacterium tuberculosis (MTB) was isolated from the culture. All the patients continued to have low dose immunosuppressive treatment, and also quadriple antituberculosis treatment [isoniazid (INH), rifampin (RIF), pyrazinamide (PRZ) and ethambutol (ETB)] has been given. The two recipients had died of disseminated form of TB. Relapse was detected in one patient 6 months after the completion of the treatment. As post-transplant TB infection develops mostly within the first year after transplantation, clinicians should be more careful for early and fast diagnosis and treatment should be started immediately.

Adult↗

Surveillance of antituberculosis drug resistance in Switzerland 1995-1997: the central link.

The purpose of the present paper is to investigate the usefulness of routine notification of antituberculosis drug susceptibilities. In Switzerland, laboratories have to report susceptibilities to isoniazid, rifampicin, ethambutol, and pyrazinamide to the Federal Office of Public Health. All clinical and laboratory information on every single tuberculosis case is routinely linked. Proportions of drug resistance were calculated and logistic regression was applied to evaluate the role of potential risk factors. Eighty percent (1056) of all culture-positive tuberculosis cases reported between October 1995 and December 1997 were analysed. The strains of 66 (6.3%) patients had resistances to at least one drug. Risk factors identified were previous antituberculosis treatment (adjusted odds ratio 7.3, 95% confidence interval 3.9-13.6), male sex (1.4, 1.1-2.0), and age <65 yrs (1.5, 1.0-2.3). Fourteen cases (1.3%), 13 of them foreign-born, were resistant to at least isoniazid and rifampicin. Reporting of drug susceptibilities allows routine assessment of the proportion of drug resistant tuberculosis and populations at risk. This proportion was found to be small in Switzerland. Risk factors were previous treatment for tuberculosis, male sex, and age <65 yrs. Resistance to at least isoniazid and rifampicin was predominantly found in foreign-born patients.

Adult↗

Therapeutic effect of DNA vaccines combined with chemotherapy in a latent infection model after aerosol infection of mice with Mycobacterium tuberculosis.

The prevention of Mycobacterium tuberculosis (M. tuberculosis) reactivation would greatly reduce the incidence of the disease, particularly among the elderly. Here, we evaluated the efficacy of DNA vaccine in combination with a conventional TB chemotherapy on the prevention of M. tuberculosis reactivation. Mice were treated with isoniazid and pyrazinamide for 3 months from 4 weeks after aerosol infection with M. tuberculosis H37Rv. During this period of chemotherapy, DNA immunization was performed three times monthly with an antigen 85A (Ag85A) DNA or an IL-12 mutant (IL-12N220L) DNA, which is known to lead to a reduction in the secretion of the p40 subunit, but not of a bioactive IL-12p70. The reactivation of M. tuberculosis was dramatically reduced in mice treated with either Ag85A DNA (P<0.01) or IL-12N220L DNA (P<0.05) in combination with chemotherapy, compared with control mice receiving only chemotherapy. Ag85A DNA vaccine showed higher IFN-gamma responses to Ag85A protein, but a lower response to culture filtrate than IL-12N220L DNA vaccine. In addition, Ag85A DNA vaccine prevented the reactivation of M. tuberculosis more efficiently than IL-12N220L DNA vaccine, indicating that Ag85A-specific IFN-gamma response might correlate with M. tuberculosis control. This study suggests that immunotherapy using Ag85A or IL-12N220L DNA vaccine combined with conventional chemotherapy might be effective clinically for the prevention of tuberculosis reactivation and may offer a more effective cure for humans than chemotherapy alone.

Animals↗

Intramedullary tuberculoma of the conus medullaris: case report and review of the literature.

OBJECTIVE: To illustrate the dilemmas in the diagnosis and management of intramedullary tuberculomas of the spinal cord. METHODS: Case report of a 32 year-old man with tuberculous meningitis. The presence of unexplained urinary retention and progressive weakness in the legs led to the discovery of an additional tuberculoma of the conus medullaris. SETTING: Dicle University Diyarbakir, Turkey. RESULTS: The patient was on a 1-year course of isoniazid, pyrazinamide and rifampicin, and responded well to conservative treatment. Our patient's unique features were represented by the worsening of neurological symptoms while being treated with adequate anti-tuberculous medication. CONCLUSION: We present a case of intramedullary tuberculoma of the conus medullaris to illustrate the dilemmas in the diagnosis and management of this curable disease, and review of the literature to date.

Adult↗

Study of the structure-activity relationships for the pyrazinamidase (PncA) from Mycobacterium tuberculosis.

In an attempt to investigate the molecular basis of pyrazinamide hydrolysis by the PncA protein from Mycobacterium tuberculosis, we determined the pyrazinamidase activity of nine PncA mutants bearing a single amino acid substitution. Among them, three mutants (D8G, K96T and S104R) had virtually no activity (< or =0.004 unit/mg), five (F13S, T61P, P69L, Y103S and A146V) retained a low level of activity (0.06-0.25 unit/mg) and one (T167L) exhibited a wild-type activity (1.51 units/mg). The possible structural effects of these substitutions were assessed by analysing a three-dimensional model of the PncA protein constructed on the basis of the crystal structure of the N-carbamoylsarcosine amidohydrolase (CSHase) from Arthrobacter sp., an amidohydrolase which was found by hydrophobic cluster analysis to be closely related to PncA. In the PncA model, five of the mutated residues, Asp-8, Phe-13, Lys-96, Tyr-103 and Ser-104, were located within a 6 A sphere around the cysteine residue Cys-138, which could be the counterpart of the active cysteine residue Cys-177 found in the CSHase. Among the remaining mutated residues, Thr-61, Pro-69 and Ala-146 were found to be more distant from Cys-138 but were associated with structural elements contributing to the catalytic centre, whereas Thr-167 was situated in an alpha-helix located far from the putative active site. These data suggest that the decrease in pyrazinamidase activity observed in the PncA mutant proteins is well correlated with the structural modifications the mutations can cause in the environment of the putative active cysteine Cys-138.

Amidohydrolases↗

Tuberculous cellulitis.

We report a case of cutaneous tuberculosis presenting as cellulitis. The patient was a 63-year-old Korean woman who also had diabetes mellitus and a 20-year-history of oral corticosteroid medication prescribed for arthralgia. In addition, she had had pulmonary tuberculosis 20-year previously for which she received systemic treatment for 1 year. Her clinical cellulitis failed to respond to antibiotic therapy. Subsequent investigations, using histopathology and polymerase chain reaction, established an alternative diagnosis of cutaneous tuberculosis. The skin eruption cleared after treatment with isoniazid, rifampicin, ethambutol and pyrazinamide. This case represents a most unusual presentation of tuberculosis in the skin. The atypical features may reflect the patient's general medical state.

Antitubercular Agents↗

Lupus vulgaris complicated by metastatic squamous cell carcinoma.

A 47-year-old Bedouin man presented with an ulcerated nodule of several months' duration on the nape of the neck. The nodule developed on an asymptomatic, slowly growing plaque which appeared during childhood. Physical examination revealed two erythematous plaques covering the posterior and right lateral aspects of the neck. The border of the plaques was soft, circinate, with a reddish-brown color, while the center was slightly erythematous and atrophic. An ulcerated nodule measuring 2 cm was seen on one of the plaques. Physical examination was unremarkable with no lymphadenopathy. Laboratory tests, including complete blood cell count, erythrocyte sedimentation rate (ESR), and routine chemistry tests, were all within normal limits. Chest X-ray showed a small calcified perihilar lymph node. The Mantoux test was positive with erythema and induration of 15 mm after 48 h. Biopsy from a plaque showed extensive diffuse granulomatous infiltration throughout the dermis with epithelioid and Langhans giant cells surrounded by mononuclear inflammatory cells. No caseation necrosis was present. Ziehl-Neelsen, periodic acid-Schiff (PAS), and Giemsa stains were negative. Polymerase chain reaction (PCR) for the detection of Mycobacterium tuberculosis and atypical mycobacteria from a skin sample was also negative. Fresh tissue cultures yielded M. tuberculosis after 6 weeks. A biopsy specimen from the ulcerated nodule demonstrated islands of atypical malignant squamous cells invading the dermis, which were compatible with moderately differentiated squamous cell carcinoma (SCC). The ulcerated nodule was completely excised, and treatment for tuberculosis was initiated with a combination of isoniazid, rifampicin, and pyrazinamide. Within 3 months of therapy, the patient's lesions resolved, leaving only slightly atrophic hypopigmented scars. A month after the treatment's initiation, an enlarged cervical lymph node was noted showing metastatic SCC on histologic examination. The patient underwent neck dissection and radiation therapy without evidence of any further metastases.

Carcinoma, Squamous Cell↗

Tuberculosis verrucosa cutis associated with tuberculous lymphadenitis.

A 34-year-old man presented with a large cutaneous lesion on his left thigh that had started as a small papule when he was 13 years of age. The lesion had enlarged slowly over the last 21 years. The patient had received bacillus Calmette-Guérin (BCG) vaccination in childhood. The family history was significant for tuberculosis. Clinical examination revealed a large, purplish-red, indurated plaque measuring 30 x 29 cm on the left thigh, extending to the buttock area. The edges of the lesion had a serpiginous contour with an involuted center (Fig. 1). A left inguinal lymph node was palpated. Chest X-ray and blood cell count were normal. No other focus of disease was identified. Laboratory testing for human immunodeficiency virus (HIV) infection was negative. Purified protein derivative (PPD) intradermal injection disclosed a 19-mm skin induration. Both the cutaneous lesion and the inguinal lymph node were biopsied. Histopathologic sections of the skin fragment showed epidermal hyperkeratosis, neovascular proliferation, and a dense dermal lymphocytic infiltrate. The histopathology of the lymph node demonstrated few granulomas with focal areas of central necrosis. Staining for fungus was negative. Ziehl-Neelsen staining was negative on both the skin and lymph node specimens. Culture for fungus and Leishmania sp. was negative. Tissue culture on Lowenstein-Jensen medium from skin and lymph node was positive for Mycobacterium colonies after 5 and 7 weeks, respectively. Multidrug therapy was instituted with rifampin 600 mg/day, isoniazid 400 mg/day, and pyrazinamide 2 g/day for 2 months, and then rifampin 600 mg/day and isoniazid 400 mg/day alone for the next 4 months. An excellent response was obtained at the end of treatment (Fig. 2).

Adult↗

Tuberculous peritonitis in 11 children: clinical features and diagnostic approach.

BACKGROUND: Tuberculous peritonitis (TBP) is a rare manifestation of childhood tuberculosis characterized by long-lasting abdominal symptoms and exudate and lymphocytes in the ascitic fluid. The diagnosis of TBP is rarely established unless a high index of suspicion is maintained. METHODS: The diagnostic features of 11 cases who were hospitalized with TBP in the Pediatric Infectious Diseases Ward of Dicle University Hospital, Turkey, were evaluated retrospectively. RESULTS: Seven cases were male and the ages of all cases ranged between 1 and 11 years. The onset of symptoms was 1-12 months (mean +/- SD 3.1 +/- 2.7 months) prior to the admission time. Nine patients gave a history of familial tuberculosis. Three cases had Bacillus Calmette-Guérin (BCG) scars and the results of five tuberculin unit (TU) tests in cases without and with BCG were over 10 and 15 mm, respectively. The most common presenting clinical symptoms and signs at admission were abdominal distention and ascites (100%), fever (27%) and loss of weight (18%). One case had accompanying tuberculous meningitis and two cases had concomitant pulmonary tuberculosis. Only one of 11 samples of ascitic fluid yielded Mycobacterium tuberculosis by the polymerase chain reaction method and no other microbiologic evidence was obtained in culture specimens. Ultrasonographic and computed tomographic imagings revealed high-density ascites that contributed well to the diagnosis. The diagnosis in two patients was proven histopathologically via peritoneoscopy and laparoscopy. All cases were treated with isoniazide, rifampisin for 9 months and pyrazinamide for the first 2 months. CONCLUSIONS: Radiologic diagnostic techniques, positive skin tests and a history of exposure to tuberculosis may contribute to the diagnosis of TBP, helped by clinical symptoms and findings, particularly when invasive diagnostic methods via peritoneoscopy and laparoscopy are not available in developing countries.

Ascites↗

Laryngeal tuberculosis in renal allograft patients.

Laryngeal tuberculosis, although the most common granulomatous disease of the larynx, is a rare form of extrapulmonary tuberculosis, never reported in immunosuppressed allograft recipients. We present two cases of laryngeal tuberculosis in renal transplant patients and a review of the literature. Two women, a 29-year-old and a 60-year-old, each more than 9 years after their cadaveric renal allograft, presented with a 2-week febrile illness with hoarseness and dysphagia, and both were found to have laryngeal tuberculosis by direct laryngoscopy. Although both radiographs were unremarkable, both patients had sputum positive for acid-fast bacilli that subsequently grew Mycobacterium tuberculosis. Clinical response promptly followed institution of isoniazid, rifampicin, and pyrazinamide in each case, although both required threefold increases in daily cyclosporin A dosage to maintain therapeutic levels.

Adult↗

Tuberculosis infection in Chinese patients undergoing continuous ambulatory peritoneal dialysis.

A retrospective study of the prevalence and pattern of tuberculosis in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) was performed. Thirty-eight cases of tuberculosis were diagnosed among 790 patients (18 men, 20 women; mean age, 58 +/- 12.6 years) between July 1994 and June 2000. The interval between the initiation of CAPD and onset of tuberculosis ranged from 1 to 168 months (median, 22 months). There were 18 cases of pulmonary tuberculosis, 14 cases of tuberculous peritonitis, 5 cases of tuberculous lymphadenitis, and 1 case of tuberculous synovitis. Patients with pulmonary tuberculosis usually presented with fever, constitutional symptoms, and pleural effusion or pulmonary infiltrates on chest radiograph. Abdominal pain and turbid dialysate were the main presenting symptoms in patients with tuberculous peritonitis. Diagnosis was established by positive culture in 20 patients, typical histological characteristics on a tissue biopsy specimen in 10 patients, and response to empirical antituberculous treatment in 8 patients. The duration of symptoms before the diagnosis of tuberculosis and initiation of antituberculous treatment ranged from 7 to 57 days (median, 30 days). Antituberculous treatment consisted of isoniazid, rifampicin, pyrazinamide, and ofloxacin for 9 to 15 months. Antituberculous treatment generally was well tolerated. Twenty-seven patients (71%) completed antituberculous treatment. No recurrence of tuberculosis was observed after a mean follow-up of 19.8 months. Eleven patients (29%) died while on antituberculous treatment; none of the deaths appeared to be directly caused by tuberculosis. We conclude that: (1) tuberculosis is prevalent among CAPD patients in our locality; (2) extrapulmonary tuberculosis, particularly tuberculous peritonitis, is common; and (3) a high index of suspicion for tuberculosis among CAPD patients is warranted to ensure early diagnosis and prompt initiation of treatment.

Aged↗

tuberculous otitis media in a renal transplant recipient.

Tuberculous otitis media is a rare cause of chronic suppurative infection of the middle ear and a very uncommon form of extrapulmonary tuberculosis. Although there have been several case reports in the nonimmunosuppressive population of tuberculous otitis media, it has never been reported in an immunosuppressed allograft recipient. We present a case of diagnosed tuberculous otitis media after recurrent chronic otitis media treated several times with empiric antibiotic treatment. After the patient developed postauricular fistula and underwent surgical removal of granulation tissue, the diagnosis was made on the basis of histopathology and growth in culture of Ziehl-Neelsen. Clinical response promptly followed institution of antituberculous treatment including isoniazid, rifampicin, ethambutol, and pyrazinamide.

Adult↗

Pancreatic mass caused by Mycobacterium tuberculosis with reduced drug sensitivity.

Pancreatic tuberculosis is a rare condition which should be considered in patients with a pancreatic mass, particularly if the patient is young, not jaundiced, from an area of high TB prevalence with a normal ERCP. We report a case of pancreatic tuberculosis due to Mycobacterium tuberculosis with reduced sensitivity to rifampicin and isoniazid, that was treated with rifabutin, ciprofloxacin, ethambutol and pyrazinamide following clinical failure of first-line therapy. The case presented illustrates the importance of obtaining material for culture and sensitivity testing in cases of suspected TB.

Adult↗

Anti-Mycobacterium tuberculosis activities of new fluoroquinolones in combination with other antituberculous drugs.

OBJECTIVES: Studies were undertaken in order to assess the anti- Mycobacterium tuberculosis (MTB) activities of newly developed fluoroquinolones in combination with other antituberculous drugs. METHODS: A new C-8-methoxyl fluoroquinolone, gatifloxacin (GFLX), and a new C-8-chloro fluoroquinolone, sitafloxacin (STFX), in combination with other drugs were examined for their activities against extracellular growing MTB organisms and those replicating in RAW264.7 macrophages (RAW-M phis s). RESULTS: STFX but not GFLX potentiated the activities of rifampin and rifalazil against extracellular MTB. Both GFLX and STFX exhibited combined activities against intramacrophage MTB, when used in combination with rifampin, rifalazil, isoniazid, pyrazinamide, ethambutol, streptomycin, or clofazimine. CONCLUSIONS: Although the observed combined effects varied to some extent from case to case depending on drug combinations, the present findings suggest the usefulness of these new fluoroquinolones in multi-drug regimens for tuberculosis patients.

Animals↗

Outcome of the treatment of culture negative tuberculosis (respiratory and non-respiratory): Blackburn 1996-2000.

OBJECTIVE: To determine the outcome of treating patients without culture confirmation of tuberculosis, with a regimen of two months of isoniazid, rifampicin, pyrazinamide and ethambutol followed by four months of isoniazid and rifampicin (2HRZE/4HR) in a setting with a rate of isoniazid resistance of 7.5% in culture confirmed cases. SETTING: Tuberculosis patients treated in the Blackburn, Hyndburn and Ribble Valley districts of the UK between 1996 and 2000 inclusive. METHODS: Patients from a detailed prospective clinical and epidemiological data base for all tuberculosis patients were studied for the years 1996-2000. RESULTS: One hundred and fourteen cases, all but two of Indian subcontinent ethnic origin, without culture confirmation had received 2HRZE/4HR. Twenty had pulmonary, 55 other respiratory and 39 non-respiratory tuberculosis. There was no bacteriologically confirmed relapse, 1 case was retreated as a clinical relapse. CONCLUSION: The treatment of tuberculosis cases without culture confirmation with a regimen of 2HRZE/4HR gives highly acceptable results. The clinical relapse rate was 0.85% (1/114), and the cure rate 99.15%.

Antitubercular Agents↗