Xpert MTB/RIF Ultra versus mycobacterial growth indicator tube liquid culture for detection of Mycobacterium tuberculosis in symptomatic adults: a diagnostic accuracy study.

BACKGROUND: Xpert MTB/RIF Ultra (Ultra) is an automated molecular test for the detection of Mycobacterium tuberculosis in sputum. We compared the sensitivity of Ultra to that of mycobacterial growth indicator tube (MGIT) liquid culture, considered the most sensitive assay in routine clinical use. METHODS: In this prospective, multicentre, cross-sectional diagnostic accuracy study, we used a non-inferiority design to assess whether the sensitivity of a single Ultra test was non-inferior to that of a single liquid culture for detection of M tuberculosis in sputum. We enrolled adults (age ≥18 years) with pulmonary tuberculosis symptoms in 11 countries and each adult provided three sputum specimens with a minimum volume of 2 mL over 2 days. Ultra was done directly on sputum 1, and Ultra and MGIT liquid culture were done on resuspended pellet from sputum 2. Results of MGIT and solid media cultures done on sputum 3 were considered the reference standard. The pre-defined non-inferiority margin was 5·0%. FINDINGS: Between Feb 18, 2016, and Dec 4, 2019, we enrolled 2906 participants. 2600 (89%) participants were analysed, including 639 (25%) of 2600 who were positive for tuberculosis by the reference standard. Of the 2357 included in the non-inferiority analysis, 877 (37%) were HIV-positive and 984 (42%) were female. Sensitivity of Ultra performed directly on sputum 1 was non-inferior to that of sputum 2 MGIT culture (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; 95% CI -2·8 to 1·1). Sensitivity of Ultra performed on sputum 2 pellet was also non-inferior to that of sputum 2 MGIT (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; -2·7 to 1·0). INTERPRETATION: For the detection of M tuberculosis in sputum from adults with respiratory symptoms, there was no difference in sensitivity of a single Ultra test to that of a single MGIT culture. Highly sensitive, rapid molecular approaches for M tuberculosis detection, combined with advances in genotypic methods for drug resistance detection, have potential to replace culture. FUNDING: US National Institute of Allergy and Infectious Diseases.

Investigating resistance in clinical Mycobacterium tuberculosis complex isolates with genomic and phenotypic antimicrobial susceptibility testing: a multicentre observational study.

BACKGROUND: Whole-genome sequencing (WGS) of Mycobacterium tuberculosis complex has become an important tool in diagnosis and management of drug-resistant tuberculosis. However, data correlating resistance genotype with quantitative phenotypic antimicrobial susceptibility testing (AST) are scarce. METHODS: In a prospective multicentre observational study, 900 clinical M tuberculosis complex isolates were collected from adults with drug-resistant tuberculosis in five high-endemic tuberculosis settings around the world (Georgia, Moldova, Peru, South Africa, and Viet Nam) between Dec 5, 2014, and Dec 12, 2017. Minimum inhibitory concentrations (MICs) and resulting binary phenotypic AST results for up to nine antituberculosis drugs were determined and correlated with resistance-conferring mutations identified by WGS. FINDINGS: Considering WHO-endorsed critical concentrations as reference, WGS had high accuracy for prediction of resistance to isoniazid (sensitivity 98·8% [95% CI 98·5-99·0]; specificity 96·6% [95% CI 95·2-97·9]), levofloxacin (sensitivity 94·8% [93·3-97·6]; specificity 97·1% [96·7-97·6]), kanamycin (sensitivity 96·1% [95·4-96·8]; specificity 95·0% [94·4-95·7]), amikacin (sensitivity 97·2% [96·4-98·1]; specificity 98·6% [98·3-98·9]), and capreomycin (sensitivity 93·1% [90·0-96·3]; specificity 98·3% [98·0-98·7]). For rifampicin, pyrazinamide, and ethambutol, the specificity of resistance prediction was suboptimal (64·0% [61·0-67·1], 83·8% [81·0-86·5], and 40·1% [37·4-42·9], respectively). Specificity for rifampicin increased to 83·9% when borderline mutations with MICs overlapping with the critical concentration were excluded. Consequently, we highlighted mutations in M tuberculosis complex isolates that are often falsely identified as susceptible by phenotypic AST, and we identified potential novel resistance-conferring mutations. INTERPRETATION: The combined analysis of mutations and quantitative phenotypes shows the potential of WGS to produce a refined interpretation of resistance, which is needed for individualised therapy, and eventually could allow differential drug dosing. However, variability of MIC data for some M tuberculosis complex isolates carrying identical mutations also reveals limitations of our understanding of the genotype and phenotype relationships (eg, including epistasis and strain genetic background). FUNDING: Bill & Melinda Gates Foundation, German Centre for Infection Research, German Research Foundation, Excellence Cluster Precision Medicine of Inflammation (EXC 2167), and Leibniz ScienceCampus EvoLUNG.

Oral Swab Specimens Tested With Xpert MTB/RIF Ultra Assay for Diagnosis of Pulmonary Tuberculosis in Children: A Diagnostic Accuracy Study.

BACKGROUND: Microbiologic diagnosis of childhood tuberculosis may be difficult. Oral swab specimens are a potential noninvasive alternative to sputum specimens for diagnosis. METHODS: This was a prospective diagnostic accuracy study of oral swab specimens (buccal and tongue) for pulmonary tuberculosis diagnosis in children (aged ≤ 15 years) in 2 South African hospital sites. Children with cough of any duration as well as a positive tuberculin skin test result, tuberculosis contact, loss of weight, or chest radiograph suggestive of pulmonary tuberculosis were enrolled. Two induced sputum specimens were tested with Xpert MTB/RIF (or Xpert MTB/RIF Ultra) assay and liquid culture. Oral swab specimens were obtained before sputum specimens, frozen, and later tested with Xpert MTB/RIF Ultra. Children were classified as microbiologically confirmed tuberculosis, unconfirmed tuberculosis (receipt of tuberculosis treatment), or unlikely tuberculosis according to National Institutes of Health consensus definitions based on sputum microbiologic results. RESULTS: Among 291 participants (median age [interquartile range], 32 [14-73] months), 57 (20%) had human immunodeficiency virus (HIV), and 87 (30%) were malnourished; 90 (31%) had confirmed pulmonary tuberculosis (rifampicin resistant in 6 [7%] ), 157 (54%), unconfirmed pulmonary tuberculosis, and 44 (15%), unlikely tuberculosis. A single oral swab specimen was obtained from 126 (43%) of the participants (tongue in 96 and buccal in 30) and 2 swab specimens from 165 (57%) (tongue in 110 and buccal in 55). Sensitivity was low (22% [95% confidence interval, 15%-32%]) for all swab specimens combined (with confirmed pulmonary tuberculosis as reference), but specificity was high (100% [91%-100%]). The highest sensitivity was 33% (95% confidence interval, 15%-58%) among participants with HIV. The overall yield was 6.9% with 1 oral swab specimen and 7.2% with 2. CONCLUSIONS: Use of the Xpert MTB/RIF Ultra assay with oral swab specimens provides poor yield for microbiologic pulmonary tuberculosis confirmation in children.

Analytical and Clinical Evaluation of the Epistem Genedrive Assay for Detection of Mycobacterium tuberculosis.

The Epistem Genedrive assay rapidly detects the Mycobacterium tuberculosis omplex from sputum and is currently available for clinical use. However, the analytical and clinical performance of this test has not been fully evaluated. The analytical limit of detection (LOD) of the Genedrive PCR amplification was tested with genomic DNA; the performance of the complete (sample processing plus amplification) system was tested by spiking M. tuberculosismc(2)6030 cells into distilled water andM. tuberculosis-negative sputum. Specificity was tested using common respiratory pathogens and nontuberculosis mycobacteria. A clinical evaluation enrolled adults with suspected pulmonary tuberculosis, obtained three sputum samples from each participant, and compared the accuracy of the Gene drive to that of the Xpert MTB/RIF assay using M. tuberculosiscultures as the reference standard. The Genedrive assay had an LOD of 1 pg/µl (100 genomic DNA copies/reaction). The LODs of the system were 2.5 × 10(4)CFU/ml and 2.5 × 10(5)CFU/ml for cells spiked into water and sputum, respectively. False-positiverpoBprobe signals were observed in 3/32 (9.4%) of the negative controls and also in few samples containing Mycobacterium abscessus,Mycobacterium gordonae, o rMycobacterium thermoresistibile In the clinical study, among 336 analyzed participants, the overall sensitivities for the tuberculosis case detection of Gene drive, Xpert, and smear microscopy were 45.4% (95% confidence interval [CI], 35.2% to 55.8%), 91.8% (95% CI, 84.4% to 96.4%), and 77.3% (95% CI, 67.7% to 85.2%), respectively. The sensitivities of Gene drive and Xpert for the detection of smear-microscopy-negative tuberculosis were 0% (95% CI, 0% to 15.4%) and 68.2% (95% CI, 45.1% to 86.1%), respectively. The Genedrive assay did not meet performance standards recommended by the World Health Organization for a smear microscopy replacement tuberculosis test. Epistem is working on modifications to improve the assay.

Impact of Xpert MTB/RIF for TB diagnosis in a primary care clinic with high TB and HIV prevalence in South Africa: a pragmatic randomised trial.

BACKGROUND: Xpert MTB/RIF is approved for use in tuberculosis (TB) and rifampicin-resistance diagnosis. However, data are limited on the impact of Xpert under routine conditions in settings with high TB burden. METHODS AND FINDINGS: A pragmatic prospective cluster-randomised trial of Xpert for all individuals with presumptive (symptomatic) TB compared to the routine diagnostic algorithm of sputum microscopy and limited use of culture was conducted in a large TB/HIV primary care clinic. The primary outcome was the proportion of bacteriologically confirmed TB cases not initiating TB treatment by 3 mo after presentation. Secondary outcomes included time to TB treatment and mortality. Unblinded randomisation occurred on a weekly basis. Xpert and smear microscopy were performed on site. Analysis was both by intention to treat (ITT) and per protocol. Between 7 September 2010 and 28 October 2011, 1,985 participants were assigned to the Xpert (n = 982) and routine (n = 1,003) diagnostic algorithms (ITT analysis); 882 received Xpert and 1,063 routine (per protocol analysis). 13% (32/257) of individuals with bacteriologically confirmed TB (smear, culture, or Xpert) did not initiate treatment by 3 mo after presentation in the Xpert arm, compared to 25% (41/167) in the routine arm (ITT analysis, risk ratio 0.51, 95% CI 0.33-0.77, p = 0.0052). The yield of bacteriologically confirmed TB cases among patients with presumptive TB was 17% (167/1,003) with routine diagnosis and 26% (257/982) with Xpert diagnosis (ITT analysis, risk ratio 1.57, 95% CI 1.32-1.87, p<0.001). This difference in diagnosis rates resulted in a higher rate of treatment initiation in the Xpert arm: 23% (229/1,003) and 28% (277/982) in the routine and Xpert arms, respectively (ITT analysis, risk ratio 1.24, 95% CI 1.06-1.44, p = 0.013). Time to treatment initiation was improved overall (ITT analysis, hazard ratio 0.76, 95% CI 0.63-0.92, p = 0.005) and among HIV-infected participants (ITT analysis, hazard ratio 0.67, 95% CI 0.53-0.85, p = 0.001). There was no difference in 6-mo mortality with Xpert versus routine diagnosis. Study limitations included incorrect intervention allocation for a high proportion of participants and that the study was conducted in a single clinic. CONCLUSIONS: These data suggest that in this routine primary care setting, use of Xpert to diagnose TB increased the number of individuals with bacteriologically confirmed TB who were treated by 3 mo and reduced time to treatment initiation, particularly among HIV-infected participants. TRIAL REGISTRATION: Pan African Clinical Trials Registry PACTR201010000255244. Please see later in the article for the Editors' Summary.

Elevated expression of the nuclear export protein, Crm1 (exportin 1), associates with human oesophageal squamous cell carcinoma.

The nuclear export receptor, Crm1 (exportin 1), is involved in the nuclear translocation of proteins and certain RNAs from the nucleus to the cytoplasm and is thus crucial for the correct localisation of cellular components. Crm1 has recently been reported to be highly expressed in certain types of cancers, yet its expression in oesophageal cancer has not been investigated to date. We investigated the expression of Crm1 in normal and tumour tissues derived from 56 patients with human oesophageal squamous cell carcinoma and its functional significance in oesophageal cancer cell line models. Immunohistochemistry revealed that Crm1 expression was significantly elevated in oesophageal tumour tissues compared to normal tissues and its localisation shifted from predominantly nuclear to nuclear and cytoplasmic. Real­time RT­PCR revealed that Crm1 expression was elevated at the mRNA level. To determine the functional significance of elevated Crm1 expression in oesophageal cancer, its expression was inhibited using siRNA, and a significant decrease in cell proliferation was observed associated with G1 cell cycle arrest and the induction of apoptosis. Similarly, leptomycin B (LMB) treatment resulted in the effective killing of oesophageal cancer cells at nanomolar concentrations. Normal oesophageal epithelial cells, however, were much less sensitive to Crm1 inhibition with siRNA and LMB. Together, this study reveals that Crm1 expression is increased in oesophageal cancer and is required for the proliferation and survival of oesophageal cancer cells.

Urine lipoarabinomannan testing for diagnosis of pulmonary tuberculosis in children: a prospective study.

BACKGROUND: Urine tests for mycobacterial lipoarabinomannan might be useful for point-of-care diagnosis of tuberculosis in adults with advanced HIV infection, but have not been assessed in children. We assessed the accuracy of urine lipoarabinomannan testing for the diagnosis of pulmonary tuberculosis in HIV-positive and HIV-negative children. METHODS: We prospectively recruited children (aged ≤ 15 years) who presented with suspected tuberculosis at a primary health-care clinic and paediatric referral hospital in South Africa, between March 1, 2009, and April 30, 2012. We assessed the diagnostic accuracy of urine lipoarabinomannan testing with lateral fl ow assay and ELISA, with mycobacterial culture of two induced sputum samples as the reference standard. Positive cultures were identified by acid-fast staining and tested to confirm Mycobacterium tuberculosis and establish susceptibility to rifampicin and isoniazid. FINDINGS: 535 children (median age 42.5 months, IQR 19.1 ­ 66.3) had urine and two induced specimens available for testing. 89 (17%) had culture-confirmed tuberculosis and 106 (20%) had HIV. The lateral fl ow lipoarabinomannan test showed poor accuracy against the reference standard, with sensitivity of 48.3% (95% CI 37.6 ­ 59.2), specificity of 60.8% (56.1 ­ 65.3), and an area under the receiver operating characteristic curve of 0.53 (0.46 ­ 0.60) for children without HIV and 0.64 (0.51 ­ 0.76) for children with HIV. ELISA had poor sensitivity in children without HIV (sensitivity 3.0%, 95% CI 0.4 ­ 10.5) and children with HIV (0%, 0.0 ­ 14.3); overall specificity was 95.7% (93.4 ­ 97.4). INTERPRETATION: Urine lipoarabinomannan tests have insufficient sensitivity and specificity to diagnose HIV-positive and HIV-negative children with tuberculosis and should not be used in this patient population.

Diagnostic accuracy of a rapid urine lipoarabinomannan test for tuberculosis in HIV-infected adults.

OBJECTIVE: In settings of high HIV prevalence, tuberculosis control and patient management are hindered by lack of accurate, rapid tuberculosis diagnostic tests that can be performed at point-of-care. The Determine TB LAM Ag (TB LAM) test is a lateral flow immunochromatographic test for detection of mycobacterial lipoarabinomannan (LAM) in urine. Our objective was to determine sensitivity and specificity of the TB LAM test for tuberculosis diagnosis. DESIGN: Prospective diagnostic accuracy study. SETTING: Hospital and outpatient settings in Uganda and South Africa. PARTICIPANTS: HIV-infected adults with tuberculosis symptoms and/or signs. METHODS: Participants provided a fresh urine specimen for TB LAM testing, blood for mycobacterial culture, and 2 respiratory specimens for smear microscopy and mycobacterial culture. MAIN OUTCOME MEASURES: For the TB LAM test, sensitivity in participants with culture-positive tuberculosis and specificity in participants without tuberculosis. RESULTS: A total of 1013 participants were enrolled. Among culture-positive tuberculosis patients, the TB LAM test identified 136/367 (37.1%) overall and 116/196 (59.2%) in the group with CD4 ≤100 cells per cubic millimeter. The test was specific in 559/573 (97.6%) patients without tuberculosis. Sensitivity of the urine TB LAM test plus sputum smear microscopy was 197/367 (53.7%) overall and 133/196 (67.9%) among those with CD4 ≤100. CD4 ≤50 [adjusted odds ratio (AOR), 6.2; P < 0.001] or 51-100 (AOR, 7.1; P < 0.001), mycobacteremia (AOR, 6.1; P < 0.01) and hospitalization (AOR, 2.6; P = 0.03) were independently associated with a positive TB LAM test. CONCLUSIONS: In HIV-positive adults with CD4 ≤100, the TB LAM urine test detected over half of culture-positive tuberculosis patients, in <30 minutes and without the need for equipment or reagents.

Xpert MTB/RIF testing of stool samples for the diagnosis of pulmonary tuberculosis in children.

In a pilot accuracy study, stool Xpert testing from 115 children with suspected pulmonary tuberculosis (PTB) detected 8/17 (47%) culture-confirmed tuberculosis cases, including 4/5 (80%) HIV-infected and 4/12 (33%) HIV-uninfected children. Sputum Xpert detected 11/17 (65%) cases. Stool holds promise for PTB diagnosis in HIV-infected children.

Rapid diagnosis of pulmonary tuberculosis in African children in a primary care setting by use of Xpert MTB/RIF on respiratory specimens: a prospective study.

BACKGROUND: In children admitted to hospital, rapid, accurate diagnosis of pulmonary tuberculosis with the Xpert MTB/RIF assay is possible, but no paediatric studies have been done in the primary care setting, where most children are given care, and where microbiological diagnosis is rarely available. We assessed the diagnostic accuracy of Xpert MTB/RIF in children in primary care. METHODS: For this prospective study, we obtained repeat induced sputum and nasopharyngeal aspirate specimens from children (<15 years) with suspected pulmonary tuberculosis at a clinic in Khayeliwtsha, Cape Town, South Africa. We compared the diagnostic accuracy of Xpert MTB/RIF with a reference standard of culture and smear microscopy on induced sputum specimens. For the main analysis, specificity of Xpert MTB/RIF versus liquid culture, we included only children with two interpretable Xpert MTB/RIF and induced sputum culture results. FINDINGS: Between Aug 1, 2010, and July 30, 2012, we enrolled 384 children (median age 38·3 months, IQR 21·2-56·5) who had one paired induced sputum and nasopharyngeal specimen, 309 (81%) of whom had two paired specimens. Five children (1%) tested positive for tuberculosis by smear microscopy, 26 (7%) tested positive by Xpert MTB/RIF, and 30 (8%) tested positive by culture. Xpert MTB/RIF on two induced sputum specimens detected 16 of 28 culture-confirmed cases (sensitivity of 57·1%, 95% CI 39·1-73·5) and on two nasopharyngeal aspirates detected 11 of 28 culture-confirmed cases (sensitivity of 39·3, 23·6-57·6; p=0·18). The specificity of Xpert MTB/RIF on induced sputum was 98·9% (95% CI 96·9-99·6) and on nasopharyngeal aspirates was 99·3% (97·4-99·8). INTERPRETATION: Our findings suggest that Xpert MTB/RIF on respiratory secretions is a useful test for rapid diagnosis of paediatric pulmonary tuberculosis in primary care. FUNDING: National Institutes of Health, National Health Laboratory Services Research Trust, the Medical Research Council of South Africa, the National Research Foundation South Africa, the European and Developing Countries Clinical Trials Partnership.

Rapid molecular diagnosis of pulmonary tuberculosis in children using nasopharyngeal specimens.

BACKGROUND: A rapid diagnosis of pediatric pulmonary tuberculosis (PTB) using Xpert MTB/RIF (Mycobacterium tuberculosis/rifampicin) automated testing on induced sputum (IS) is possible, but the capacity for performing IS is limited. The diagnosis using a nasopharyngeal aspirate (NPA), which can be non-invasively obtained, is desirable. METHODS: Paired specimens (NPA and IS) were tested using smear, liquid culture and Xpert. The diagnostic accuracy of Xpert and smear was compared with culture for different specimens in children with suspected PTB. RESULTS: There were 535 children [median age 19 months, 117 (21·9%) HIV-infected] who had one IS and one NPA specimen; 396 had two paired specimens. A positive smear, Xpert test or culture occurred in 30 (5.6%), 81 (15.1%) and 87 children (16.3%), respectively. The culture yield was higher from IS (84/87, 96.6%) vs NPA (61/87, 70.1%, P < .001). Amongst children with two paired specimens, 63 culture-confirmed cases occurred [60 (95.2%) IS vs 48 (76.2%) NPA, P = .002]. The sensitivity of two Xpert tests was similar for IS and NPAs [(45/63) 71% vs (41/63) 65%, P = .444)]; the sensitivity of smear was lower for IS (21/63, 33%) and NPA (16/63, 25%). The incremental yield from a second IS was 9 cases (17.6%) by culture and 9 (25%) by Xpert testing; a second NPA increased the culture yield by 10 (26.3%) and Xpert by 11 (36.7%). Xpert specificity was 99.1% (98.1-100) for IS and 98.2% (96.8-99.6) for NPAs. Xpert testing provided faster results than culture (median 0 vs 15 days, P < .001). CONCLUSIONS: Xpert testing on 2 NPAs is useful in children with suspected PTB, particularly in settings where IS and culture are not feasible.

Accuracy of the Xpert MTB/RIF test for the diagnosis of pulmonary tuberculosis in children admitted to hospital in Cape Town, South Africa: a descriptive study.

BACKGROUND: WHO recommends that Xpert MTB/RIF replaces smear microscopy for initial diagnosis of suspected HIV-associated tuberculosis or multidrug-resistant pulmonary tuberculosis, but no data exist for its use in children. We aimed to assess the accuracy of the test for the diagnosis of pulmonary tuberculosis in children in an area with high tuberculosis and HIV prevalences. METHODS: In this prospective, descriptive study, we enrolled children aged 15 years or younger who had been admitted to one of two hospitals in Cape Town, South Africa, with suspected pulmonary tuberculosis between Feb 19, 2009, and Nov 30, 2010. We compared the diagnostic accuracy of MTB/RIF and concentrated, fluorescent acid-fast smear with a reference standard of liquid culture from two sequential induced sputum specimens (primary analysis). RESULTS: 452 children (median age 19·4 months, IQR 11·1-46·2) had at least one induced sputum specimen; 108 children (24%) had HIV infection. 27 children (6%) had a positive smear result, 70 (16%) had a positive culture result, and 58 (13%) had a positive MTB/RIF test result. With mycobacterial culture as the reference standard, MTB/RIF tests when done on two induced sputum samples detected twice as many cases (75·9%, 95% CI 64·5-87·2) as did smear microscopy (37·9%, 25·1-50·8), detecting all of 22 smear-positive cases and 22 of 36 (61·1%, 44·4-77·8) smear-negative cases. For smear-negative cases, the incremental increase in sensitivity from testing a second specimen was 27·8% for MTB/RIF, compared with 13·8% for culture. The specificity of MTB/RIF was 98·8% (97·6-99·9). MTB/RIF results were available in median 1 day (IQR 0-4) compared with median 12 days (9-17) for culture (p<0·0001). INTERPRETATION: MTB/RIF testing of two induced sputum specimens is warranted as the first-line diagnostic test for children with suspected pulmonary tuberculosis. FUNDING: National Institutes of Health, the National Health Laboratory Service Research Trust, the Medical Research Council of South Africa, and Wellcome Trust.