SummaryBackgroundThe Xpert MTB/RIF assay is an automated molecular test that has improved the detection of tuberculosis and rifampicin resistance, but its sensitivity is inadequate in patients with paucibacillary disease or HIV. Xpert MTB/RIF Ultra (Xpert Ultra) was developed to overcome this limitation. We compared the diagnostic performance of Xpert Ultra with that of Xpert for detection of tuberculosis and rifampicin resistance.MethodsIn this prospective, multicentre, diagnostic accuracy study, we recruited adults with pulmonary tuberculosis symptoms presenting at primary health-care centres and hospitals in eight countries (South Africa, Uganda, Kenya, India, China, Georgia, Belarus, and Brazil). Participants were allocated to the case detection group if no drugs had been taken for tuberculosis in the past 6 months or to the multidrug-resistance risk group if drugs for tuberculosis had been taken in the past 6 months, but drug resistance was suspected. Demographic information, medical history, chest imaging results, and HIV test results were recorded at enrolment, and each participant gave at least three sputum specimen on 2 separate days. Xpert and Xpert Ultra diagnostic performance in the same sputum specimen was compared with culture tests and drug susceptibility testing as reference standards. The primary objectives were to estimate and compare the sensitivity of Xpert Ultra test with that of Xpert for detection of smear-negative tuberculosis and rifampicin resistance and to estimate and compare Xpert Ultra and Xpert specificities for detection of rifampicin resistance. Study participants in the case detection group were included in all analyses, whereas participants in the multidrug-resistance risk group were only included in analyses of rifampicin-resistance detection.FindingsBetween Feb 18, and Dec 24, 2016, we enrolled 2368 participants for sputum sampling. 248 participants were excluded from the analysis, and 1753 participants were distributed to the case detection group (n=1439) and the multidrug-resistance risk group (n=314). Sensitivities of Xpert Ultra and Xpert were 63% and 46%, respectively, for the 137 participants with smear-negative and culture-positive sputum (difference of 17%, 95% CI 10 to 24); 90% and 77%, respectively, for the 115 HIV-positive participants with culture-positive sputum (13%, 6·4 to 21); and 88% and 83%, respectively, across all 462 participants with culture-positive sputum (5·4%, 3·3 to 8·0). Specificities of Xpert Ultra and Xpert for case detection were 96% and 98% (−2·7%, −3·9 to −1·7) overall, and 93% and 98% for patients with a history of tuberculosis. Xpert Ultra and Xpert performed similarly in detecting rifampicin resistance.InterpretationFor tuberculosis case detection, sensitivity of Xpert Ultra was superior to that of Xpert in patients with paucibacillary disease and in patients with HIV. However, this increase in sensitivity came at the expense of a decrease in specificity.FundingGovernment of Netherlands, Government of Australia, Bill & Melinda Gates Foundati...

Multidrug-resistant tuberculosis (MDR-TB), caused by drug resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. In this study, we examined a dataset of 5,310 M. tuberculosis whole genome sequences from five continents. Despite great diversity with respect to geographic point of isolation, genetic background and drug resistance, patterns of drug resistance emergence were conserved globally. We have identified harbinger mutations that often precede MDR. In particular, the katG S315T mutation, conferring resistance to isoniazid, overwhelmingly arose before rifampicin resistance across all lineages, geographic regions, and time periods. Molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of pre-MDR polymorphisms, particularly katG S315, into molecular diagnostics will enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

Background Repeat COVID-19 molecular testing can lead to positive test results after negative tests and to multiple positive test results over time. The association between positive tests and infectious virus is important to quantify. Methods A two months cohort of retrospective data and consecutively collected specimens from COVID-19 patients or patients under investigation were used to understand the correlation between prolonged viral RNA positive test results, cycle threshold (Ct) values and growth of SARS-CoV-2 in cell culture. Whole genome sequencing was used to confirm virus genotype in patients with prolonged viral RNA detection. Droplet digital PCR (ddPCR) was used to assess the rate of false negative COVID-19 diagnostic tests. Results In two months, 29,686 specimens were tested and 2,194 patients received repeated testing. Virus recovery in cell culture was noted in specimens with SARS-CoV-2 target genes’ Ct value average of 18.8 ± 3.4. Prolonged viral RNA shedding was associated with positive virus growth in culture in specimens collected up to 20 days after the first positive result but mostly in individuals symptomatic at time of sample collection. Whole genome sequencing provided evidence the same virus was carried over time. Positive tests following negative tests had Ct values higher than 29.5 and were not associated with virus culture. ddPCR was positive in 5.6% of negative specimens collected from COVID-19 confirmed or clinically suspected patients. Conclusions Low Ct values in SARS-CoV-2 diagnostic tests were associated with virus growth in cell culture. Symptomatic patients with prolonged viral RNA shedding can also be infectious.

Background Symptom-based screening for tuberculosis (TB) is recommended for all people living with HIV (PLHIV) resulting in unnecessary Xpert MTB/RIF testing for the vast majority of individuals living in TB endemic areas and thus, poor implementation of intensified case finding (ICF) and TB preventive therapy. Novel approaches to TB screening are therefore critical in achieving global targets for TB elimination. Methods In a prospective study of PLHIV with CD4+ T-cell count ≤350 cells/uL initiating antiretroviral therapy (ART) from two HIV/AIDS clinics in Uganda, we evaluated the performance of C-reactive protein (CRP) measured using a rapid and inexpensive point-of-care (POC) assay as a screening tool for active pulmonary TB. Findings Of 1177 HIV-infected adults (median CD4+ T-cell count 168 cells/µL) enrolled, 163 (14%) had culture-confirmed TB. POC CRP had 89% (145/163) sensitivity and 72% (731/1014) specificity for culture-confirmed TB. Compared to the WHO symptom screen, POC CRP had lower sensitivity (difference −7% [95% CI: −12 to −2], p=0.002) but substantially higher specificity (difference +58% [95% CI: +61 to +55], p<0.0001). When Xpert MTB/RIF results were used as the reference standard, sensitivity of POC CRP and the WHO symptom screen were similar (94% [79/84] vs. 99% [83/84]; difference −5% [95% CI: −12 to +2], p=0.10). Interpretation The performance characteristics of CRP support its use as a TB screening test for PLHIV with CD4+ T-cell count ≤350 cells/µL initiating ART. HIV/AIDS programs should consider POC CRP-based TB screening to improve the efficiency of ICF and increase uptake of TB preventive therapy. FUNDING National Institutes of Health; Presidential Emergency Plan for AIDS Relief; University of California, San Francisco, Nina Ireland Program for Lung Health

f For Mycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first-and second-line antituberculosis drugs. To evaluate the performance of MYCOTB for M. tuberculosis drug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archived M. tuberculosis isolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was >92% for 7 of 12 drugs when a strict definition was used and >96% for 10 of 12 drugs when agreement was defined by allowing a ؎ one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was >95% for all drugs. MYCOTB provided reliable results for M. tuberculosis susceptibility testing of first-and second-line drugs except ethambutol, and results were available sooner than those determined by APM.T he emergence and spread of drug-resistant strains of Mycobacterium tuberculosis comprise a serious threat to tuberculosis (TB) control (1, 2). Knowledge of M. tuberculosis drug susceptibility is important in optimizing individual patient management and TB control in populations. Genotypic methods have the potential for a very short time to results, but to date, the knowledge of the full spectrum of genetic loci and mutations associated with resistance to many antituberculosis drugs is incomplete (3,4,5,6,7). Phenotypic methods therefore remain important. The reference phenotypic method-the indirect agar proportion method (APM) using Middlebrook solid media-is qualitative and based on drug critical concentrations. Limitations of the APM and related methods include lack of standardization and in some cases the need for in-laboratory preparation of drug stocks and agar plates, which can be a source of variability over time and between laboratories. Critical concentrations are based on historical epidemiological data and for some drugs are not well-aligned with achievable drug serum concentrations or accurate in predicting clinical failure (8,9,10). Studies on molecular drug resistance mechanisms in M. tuberculosis have shown that, at least for some antibiotics, different mutations are associated with different MICs, furthe...

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 two 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 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/mm3. The test was specific in 559/573 (97.6%) of 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 less than 30 minutes and without the need for equipment or reagents.

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.)

Background Outpatient COVID-19 has been insufficiently characterized. To determine the progression of disease and determinants of hospitalization, we conducted a prospective cohort study. Methods Outpatient adults with positive RT-PCR results for SARS-CoV-2 were recruited by phone between April 21 to July 23, 2020 after receiving outpatient or emergency department testing within a large health network in Maryland, USA. Symptoms were collected by participants on days 0, 3, 7, 14, 21, and 28 and portable pulse oximeter oxygen saturation (SaO2), heart rate, and temperature were collected for 15 consecutive days. Baseline demographics, comorbid conditions, and vital signs were evaluated for risk of subsequent hospitalization using negative binomial, and logistic regression. Results Among 118 SARS-CoV-2 infected outpatients, the median age was 56.0 years (IQR, 50.0 to 63.0) and 50 (42.4%) were male. Among individuals in the first week of illness (N=61), the most common symptoms included weakness/fatigue (65.7%), cough (58.8%), headache (45.6%), chills (38.2%), and anosmia (27.9%). Participants returned to their usual health a median of 20 days (IQR, 13 to 38) from symptom onset, and 66.0% of respondents were at their usual health during the fourth week of illness. Over 28 days, 10.9% presented to the emergency department and 7.6% required hospitalization. The area under the receiving operating characteristic curve for the initial home SaO2 for predicting subsequent hospitalization was 0.86 (CI, 0.73 to 0.99). Conclusions Symptoms often persisted but uncommonly progressed to hospitalization among outpatients with COVID-19. Home SaO2 may be a helpful tool to stratify risk of hospitalization.