Efficacy of Xpert in tuberculosis diagnosis based on various specimens: a systematic review and meta-analysis.

Objective: The GeneXpert MTB/RIF assay (Xpert) is a diagnostic tool that has been shown to significantly improve the accuracy of tuberculosis (TB) detection in clinical settings, with advanced sensitivity and specificity. Early detection of TB can be challenging, but Xpert has improved the efficacy of the diagnostic process. Nevertheless, the accuracy of Xpert varies according to different diagnostic specimens and TB infection sites. Therefore, the selection of adequate specimens is critical when using Xpert to identify suspected TB. As such, we have conducted a meta-analysis to evaluate the effectiveness of Xpert for diagnosis of different TB types using several specimens. Methods: We conducted a comprehensive search of several electronic databases, including PubMed, Embase, the Cochrane Central Register of Controlled Trials, and the World Health Organization clinical trials registry center, covering studies published from Jan 2008 to July 2022. Data were extracted using an adapted version of the Checklist for Critical Appraisal and Data Extraction for Systematic Reviews of Prediction Modeling Studies. Where appropriate, meta-analysis was performed using random-effects models. The risk of bias and level of evidence was assessed using the Quality in Prognosis Studies tool and a modified version of the Grading of Recommendations Assessment, Development, and Evaluation. RStudio was utilized to analyze the results, employing the meta4diag, robvis, and metafor packages. Results: After excluding duplicates, a total of 2163 studies were identified, and ultimately, 144 studies from 107 articles were included in the meta-analysis based on predetermined inclusion and exclusion criteria. Sensitivity, specificity and diagnostic accuracy were estimated for various specimens and TB types. In the case of pulmonary TB, Xpert using sputum (0.95 95%CI 0.91-0.98) and gastric juice (0.94 95%CI 0.84-0.99) demonstrated similarly high sensitivity, surpassing other specimen types. Additionally, Xpert exhibited high specificity for detecting TB across all specimen types. For bone and joint TB, Xpert, based on both biopsy and joint fluid specimens, demonstrated high accuracy in TB detection. Furthermore, Xpert effectively detected unclassified extrapulmonary TB and tuberculosis lymphadenitis. However, the Xpert accuracy was not satisfactory to distinguish TB meningitis, tuberculous pleuritis and unclassified TB. Conclusions: Xpert has exhibited satisfactory diagnostic accuracy for most TB infections, but the efficacy of detection may vary depending on the specimens analyzed. Therefore, selecting appropriate specimens for Xpert analysis is essential, as using inadequate specimens can reduce the ability to distinguish TB. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=370111, identifier CRD42022370111.

Analysis of serum microRNA-122 in a randomized controlled trial of N-acetylcysteine for treatment of antituberculosis drug-induced liver injury.

AIM: Serum microRNA-122 (miR-122) is a novel biomarker for drug-induced liver injury, with good sensitivity in the early diagnosis of paracetamol-induced liver injury. We describe miR-122 concentrations in participants with antituberculosis drug-induced liver injury (AT-DILI). We explored the relationship between miR-122 and alanine aminotransferase (ALT) concentrations and the effect of N-acetylcysteine (NAC) on miR-122 concentrations. METHODS: We included participants from a randomized placebo-controlled trial of intravenous NAC in AT-DILI. ALT and miR-122 concentrations were quantified before and after infusion of NAC/placebo. We assessed correlations between ALT and miR-122 concentrations and described changes in ALT and miR-122 concentrations between sampling occasions. RESULTS: We included 45 participants; mean age (± standard deviation) 38 (±10) years, 58% female and 91% HIV positive. The median (interquartile range) time between pre- and post-infusion biomarker specimens was 68 h (47-77 h). The median pre-infusion ALT and miR-122 concentrations were 420 U/L (238-580) and 0.58 pM (0.18-1.47), respectively. Pre-infusion ALT and miR-122 concentrations were correlated (Spearman's ρ = .54, P = .0001). Median fold-changes in ALT and miR-122 concentrations between sampling were 0.56 (0.43-0.69) and 0.75 (0.23-1.53), respectively, and were similar in the NAC and placebo groups (P = .40 and P = .68 respectively). CONCLUSIONS: miR-122 concentrations in our participants with AT-DILI were considerably higher than previously reported in healthy volunteers and in patients on antituberculosis therapy without liver injury. We did not detect an effect of NAC on miR-122 concentrations. Further research is needed to determine the utility of miR-122 in the diagnosis and management of AT-DILI.

Xpert MTB/RIF Ultra in the auxiliary diagnosis of tuberculosis among people living with human immunodeficiency virus.

Clinical diagnosis of tuberculosis (TB) in people living with the human immunodeficiency virus (HIV) poses a challenge. The Xpert MTB/RIF Ultra (Ultra) has displayed greater sensitivity at diagnosing tuberculosis and rifampicin resistance compared to the Xpert MTB/RIF (Xpert). However, whether Ultra is able to facilitate an auxiliary diagnosis of TB in patients with an HIV-TB co-infection remains unclear. Accordingly, the current study evaluated the use of Ultra in patients with an HIV-TB co-infection by summarizing relevant studies. The sensitivity and specificity of Ultra and Xpert at diagnosing patients with an HIV-TB co-infection have been summarized and compared. The performance of Ultra in diagnosing extrapulmonary tuberculosis was also summarized. Although a large-cohort, multi-center study needs to be conducted to assess Ultra's ability to detect TB in AIDS patients in the future, the current evidence supports the use of Ultra for the assessment of patients with an HIV-TB co-infection.

Xpert MTB/RIF Ultra assay for tuberculosis disease and rifampicin resistance in children.

BACKGROUND: Every year, an estimated one million children and young adolescents become ill with tuberculosis, and around 226,000 of those children die. Xpert MTB/RIF Ultra (Xpert Ultra) is a molecular World Health Organization (WHO)-recommended rapid diagnostic test that simultaneously detects Mycobacterium tuberculosis complex and rifampicin resistance. We previously published a Cochrane Review 'Xpert MTB/RIF and Xpert MTB/RIF Ultra assays for tuberculosis disease and rifampicin resistance in children'. The current review updates evidence on the diagnostic accuracy of Xpert Ultra in children presumed to have tuberculosis disease. Parts of this review update informed the 2022 WHO updated guidance on management of tuberculosis in children and adolescents. OBJECTIVES: To assess the diagnostic accuracy of Xpert Ultra for detecting: pulmonary tuberculosis, tuberculous meningitis, lymph node tuberculosis, and rifampicin resistance, in children with presumed tuberculosis. Secondary objectives To investigate potential sources of heterogeneity in accuracy estimates. For detection of tuberculosis, we considered age, comorbidity (HIV, severe pneumonia, and severe malnutrition), and specimen type as potential sources. To summarize the frequency of Xpert Ultra trace results. SEARCH METHODS: We searched the Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, three other databases, and three trial registers without language restrictions to 9 March 2021. SELECTION CRITERIA: Cross-sectional and cohort studies and randomized trials that evaluated Xpert Ultra in HIV-positive and HIV-negative children under 15 years of age. We included ongoing studies that helped us address the review objectives. We included studies evaluating sputum, gastric, stool, or nasopharyngeal specimens (pulmonary tuberculosis), cerebrospinal fluid (tuberculous meningitis), and fine needle aspirate or surgical biopsy tissue (lymph node tuberculosis). For detecting tuberculosis, reference standards were microbiological (culture) or composite reference standard; for stool, we also included Xpert Ultra performed on a routine respiratory specimen. For detecting rifampicin resistance, reference standards were drug susceptibility testing or MTBDRplus. DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and, using QUADAS-2, assessed methodological quality judging risk of bias separately for each target condition and reference standard. For each target condition, we used the bivariate model to estimate summary sensitivity and specificity with 95% confidence intervals (CIs). We stratified all analyses by type of reference standard. We summarized the frequency of Xpert Ultra trace results; trace represents detection of a very low quantity of Mycobacterium tuberculosis DNA. We assessed certainty of evidence using GRADE. MAIN RESULTS: We identified 14 studies (11 new studies since the previous review). For detection of pulmonary tuberculosis, 335 data sets (25,937 participants) were available for analysis. We did not identify any studies that evaluated Xpert Ultra accuracy for tuberculous meningitis or lymph node tuberculosis. Three studies evaluated Xpert Ultra for detection of rifampicin resistance. Ten studies (71%) took place in countries with a high tuberculosis burden based on WHO classification. Overall, risk of bias was low. Detection of pulmonary tuberculosis Sputum, 5 studies Xpert Ultra summary sensitivity verified by culture was 75.3% (95% CI 64.3 to 83.8; 127 participants; high-certainty evidence), and specificity was 97.1% (95% CI 94.7 to 98.5; 1054 participants; high-certainty evidence). Gastric aspirate, 7 studies Xpert Ultra summary sensitivity verified by culture was 70.4% (95% CI 53.9 to 82.9; 120 participants; moderate-certainty evidence), and specificity was 94.1% (95% CI 84.8 to 97.8; 870 participants; moderate-certainty evidence). Stool, 6 studies Xpert Ultra summary sensitivity verified by culture was 56.1% (95% CI 39.1 to 71.7; 200 participants; moderate-certainty evidence), and specificity was 98.0% (95% CI 93.3 to 99.4; 1232 participants; high certainty-evidence). Nasopharyngeal aspirate, 4 studies Xpert Ultra summary sensitivity verified by culture was 43.7% (95% CI 26.7 to 62.2; 46 participants; very low-certainty evidence), and specificity was 97.5% (95% CI 93.6 to 99.0; 489 participants; high-certainty evidence). Xpert Ultra sensitivity was lower against a composite than a culture reference standard for all specimen types other than nasopharyngeal aspirate, while specificity was similar against both reference standards. Interpretation of results In theory, for a population of 1000 children: • where 100 have pulmonary tuberculosis in sputum (by culture): - 101 would be Xpert Ultra-positive, and of these, 26 (26%) would not have pulmonary tuberculosis (false positive); and - 899 would be Xpert Ultra-negative, and of these, 25 (3%) would have tuberculosis (false negative). • where 100 have pulmonary tuberculosis in gastric aspirate (by culture): - 123 would be Xpert Ultra-positive, and of these, 53 (43%) would not have pulmonary tuberculosis (false positive); and - 877 would be Xpert Ultra-negative, and of these, 30 (3%) would have tuberculosis (false negative). • where 100 have pulmonary tuberculosis in stool (by culture): - 74 would be Xpert Ultra-positive, and of these, 18 (24%) would not have pulmonary tuberculosis (false positive); and - 926 would be Xpert Ultra-negative, and of these, 44 (5%) would have tuberculosis (false negative). • where 100 have pulmonary tuberculosis in nasopharyngeal aspirate (by culture): - 66 would be Xpert Ultra-positive, and of these, 22 (33%) would not have pulmonary tuberculosis (false positive); and - 934 would be Xpert Ultra-negative, and of these, 56 (6%) would have tuberculosis (false negative). Detection of rifampicin resistance Xpert Ultra sensitivity was 100% (3 studies, 3 participants; very low-certainty evidence), and specificity range was 97% to 100% (3 studies, 128 participants; low-certainty evidence). Trace results Xpert Ultra trace results, regarded as positive in children by WHO standards, were common. Xpert Ultra specificity remained high in children, despite the frequency of trace results. AUTHORS' CONCLUSIONS: We found Xpert Ultra sensitivity to vary by specimen type, with sputum having the highest sensitivity, followed by gastric aspirate and stool. Nasopharyngeal aspirate had the lowest sensitivity. Xpert Ultra specificity was high against both microbiological and composite reference standards. However, the evidence base is still limited, and findings may be imprecise and vary by study setting. Although we found Xpert Ultra accurate for detection of rifampicin resistance, results were based on a very small number of studies that included only three children with rifampicin resistance. Therefore, findings should be interpreted with caution. Our findings provide support for the use of Xpert Ultra as an initial rapid molecular diagnostic in children being evaluated for tuberculosis.

Xpert MTB/RIF Ultra outperformed the Xpert assay in tuberculosis lymphadenitis diagnosis: a prospective head-to-head cohort study.

OBJECTIVES: Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF) Ultra (Xpert-Ultra) has shown better sensitivity in comparison with Xpert MTB/RIF (Xpert) in extrapulmonary tuberculosis (TB), whereas the head-to-head comparison of these methods in TB lymphadenitis had barely been performed. METHODS: Patients with undiagnosed lymphadenopathy were recruited prospectively and consecutively, and fine-needle aspiration (FNA) biopsy or lymph node tissue was collected. The specimen was subjected to smear, culture, Xpert, and Xpert-Ultra assays. Culture and/or smear for acid-fast bacilli (AFB) or AFB observed on histopathology were performed as a reference. RESULTS: A total of 106 participants were recruited, including 41 confirmed TB, 33 probable TB, and 32 non-TB lymphadenopathies. The head-to-head comparison for MTB detection showed that Xpert-Ultra produced the highest sensitivity when compared with smear, culture, and Xpert (75.7% vs 5.4 %, 13.5%, and 48.7%). When Xpert-Ultra outcomes were integrated for diagnosis, the percentage of confirmed TB lymphadenitis cases increased from 55.4% (41/74) to 85.1% (63/74). The sensitivities of Xpert-Ultra and Xpert on tissue were 73.6% (95% CI: 59.4-84.3) and 39.6% (95% CI: 26.8-54.0), respectively. The sensitivity of Xpert-Ultra on FNA samples (81.0%, 95% CI: 57.4-93.7) was higher than that of Xpert (71.4%, 95% CI: 47.7-87.8). CONCLUSION: Xpert-Ultra detected significantly more TB lymphadenitis cases than Xpert or culture. This superiority was particularly distinct using lymph node tissue than FNA detection.

Evaluating the efficacy of stool sample on Xpert MTB/RIF Ultra and its comparison with other sample types by meta-analysis for TB diagnostics.

Precise and timely detection of tuberculosis (TB) is crucial to reduce transmission. This study aims to assess the accuracy of Xpert MTB/RIF Ultra on stool samples and systematically review the performance of Xpert MTB/RIF Ultra with different sample types by meta-analysis. Stool samples of smear-negative pulmonary TB (PTB), cervical lymph node TB, and abdominal TB patients were tested on the Xpert MTB/RIF Ultra system. Meta-analysis was performed on a set of 44 studies. Data were grouped by sample type, and the pooled sensitivity and specificity of Xpert MTB/RIF Ultra were calculated. The sensitivity of Xpert MTB/RIF Ultra with stool samples was 100% for smear-negative PTB, 27.27% for cervical lymph node TB, and 50% for abdominal TB patients, with 100% specificity for all included TB groups. The summary estimate for all PTB samples showed 84.2% sensitivity and 94.5% specificity, and EPTB samples showed 88.6% sensitivity and 96.4% specificity. Among all sample types included in our meta-analysis, urine showed the best performance for EPTB diagnosis. This pilot study supports the use of stool as an alternative non-invasive sample on Xpert MTB/RIF Ultra for rapid testing, suitable for both PTB and EPTB diagnosis.

Evaluating diagnostic performance of Truenat MTB Plus for gastrointestinal tuberculosis.

BACKGROUND AND AIM: Prompt and accurate diagnosis of gastrointestinal tuberculosis (GITB) along with simultaneous detection of drug resistance is inevitable for tuberculosis elimination. Truenat MTB Plus (TruPlus), a chip-based real-time polymerase chain reaction assay, was evaluated for the first time for diagnosing GITB and detecting rifampicin resistance. METHODS: Fifty ileocecal biopsy specimens (5 microbiologically confirmed GITB [culture-positive], 25 clinically confirmed GITB [culture-negative], and 20 control patients) processed in the Department of Microbiology between 2011 and 2021 were subjected to TruPlus assay, Xpert MTB RIF assay multiplex polymerase chain reaction. Their performance was evaluated against both culture and composite reference standard. RESULTS: The overall sensitivity and specificity of TruPlus in diagnosing GITB was 70% (21/30) and 100%, respectively. The sensitivity was 60% (3/5) for microbiologically confirmed cases and 72% (18/25) for clinically confirmed cases. Performance of TruPlus was superior to Xpert (sensitivity = 30%; P = 0.001) and comparable with MPCR (sensitivity = 83.33%; P = 0.13). Both TruPlus and MPCR had moderate agreement with reference standards, and MPCR detected additional three cases. Both TruPlus and Xpert correctly reported Rifampicin resistance in three cases. CONCLUSIONS: TruPlus, with its greater portability and higher sensitivity than Xpert, could serve as an important tool for diagnosing GITB and rifampicin resistance at outreach endemic areas.

A cost-benefit algorithm for rapid diagnosis of tuberculosis and rifampicin resistance detection during mass screening campaigns.

BACKGROUND: Active tuberculosis (TB) case finding is important as it helps detect pulmonary TB cases missed by the other active screening methods. It requires periodic mass screening in risk population groups such as prisoners and refugees. Unfortunately, in these risk population groups periodic mass screening can be challenging due to lengthy turnaround time (TAT), cost and implementation constraints. The aim of this study was to evaluate a diagnostic algorithm that can reduce the TAT and cost for TB and Rifampicin resistance (RR) detection. The algorithm involves testing with TB-LAMP followed by Xpert MTB/RIF for positive TB-LAMP cases to diagnose TB during mass campaigns in prisons and refugee camps. METHODS: The National Tuberculosis Control Program (NTCP) organized routine TB mass-screening campaigns in 34 prisons and 3 villages with refugees camps in Cameroon in 2019. TB LAMP was used for initial TB diagnosis and all TB-LAMP positive cases tested with the Xpert MTB/RIF assay to determine RR. TAT and cost benefits analysis of the combined use of TB-LAMP and Xpert MTB/RIF assays was determined and compared to the Xpert MTB/RIF assay when used only. RESULTS: A total of 4075 sputum samples were collected from TB presumptive, 3672 cases in 34 prisons and 403 samples in 3 villages. Of the 4,075 samples screened with TB-LAMP, 135 were TB positive (3.31%) and run on the Xpert MTB/RIF. Of the 135 positives cases, Xpert MTB/RIF revealed 3 were RR (2.22%). The use of TB-LAMP followed by testing with Xpert MTB/RIF for TB and RR detection reduced the TAT by 73.23% in prisons and 74.92% in villages. In addition to a reduced TAT, the two molecular tests used in synergy is cost benefit from year 2 onwards. CONCLUSION: This study demonstrates the advantages of a diagnostic algorithm based on an initial testing with TB-LAMP followed by testing with Xpert MTB/RIF for TB diagnosis. This approach improved early and rapid TB detection with an added advantage of providing RR status. The proposed algorithm is effective and less costly from the second year of implementation and should be used by TB control programs.

Protective Effect of Rifampicin Loaded by HPMA-PLA Nanopolymer on Macrophages Infected with Mycobacterium Tuberculosis.

PURPOSE: This research was designed to investigate the protective effect of rifampicin (RIF) loaded by N-(2-hydroxypropyl) methylacrylamide- (HPMA-) polylactic acid (PLA) nanopolymer on macrophages infected with Mycobacterium tuberculosis (MTB). METHODS: We first induced H37Rv to infect macrophages to build a cell model. Then, the HPMA-PLA nanopolymer loaded with RIF was prepared to treat MTB-infected macrophages. The macrophage activity was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the nitric oxide (NO) in cells was measured through Griess reagent, and the bacterial activity of MTB was observed via the colony-forming unit (CFU) assay. The inflammation-related factors in cells were detected via the enzyme-linked immunosorbent assay (ELISA), the apoptosis of macrophages was examined via flow cytometry, and the expression of apoptosis-related proteins was determined by western blot (WB). RESULTS: HPMA-PLA had no obvious toxicity to macrophages. The expression of NO and inflammatory factors in macrophages infected with MTB increased significantly, but the apoptosis rate was not significantly different from that of uninfected cells. However, after treatment with HPMA-PLA-RIF or free RIF, the inflammatory reaction of infected cells was inhibited, the expression of NO was decreased, the apoptosis rate was increased, and the bacterial activity in cells was decreased, with statistically significant differences; moreover, HPMA-PLA-RIF was more effective than free RIF. CONCLUSIONS: HPMA-PLA-RIF has a high protective effect on macrophages infected with MTB, with high safety. Its protective mechanism is at least partly through inhibiting the production of NO and inflammatory response, which can inhibit bacterial activity and induce cell apoptosis.

The Great Masquerader - TB Osteoarthritis.

OBJECTIVES: TB arthritis is a rarely reported entity in Western literature and its ability to masquerade as many other diseases makes it difficult to diagnose. We report an interesting case of TB arthritis of the ankle. METHODS: We present a 44 year-old diabetic Chinese male with a recent history of worsening pain, swelling, and redness in his left foot with an abscess and X-ray findings consistent with Charcot foot. RESULTS: At first, the presentation was believed to be Charcot's foot with MSSA osteomyelitis but after the wound culture and bone biopsy were both positive for Mycobacterium tuberculosis as well, the diagnosis of tuberculous arthritis was confirmed. CONCLUSIONS: While the prevalence of TB and other diseases is low in the majority of the United States, we still need to be aware of such diseases in populations with increasing migration and be cognizant of the potential impact of a patient's background on a diagnosis is critical to properly diagnosing and treating patients. Vascular surgeons may be seeing patients with abscesses of the lower extremities and may miss the diagnosis if cultures for TB are not sought.

Application of loop-mediated isothermal amplification and next-generation sequencing in the diagnosis of maternal tuberculosis with multiple negative tests: A case report.

RATIONALE: Tuberculosis (TB) is one of the top 10 causes of death worldwide and is the leading infectious cause of death. The incidence of TB, especially active TB, is increased in pregnant and postpartum women. Newborns can be infected with TB from their mothers through several routes. Diagnosis of TB in pregnant women and infants is difficult. Here, we report the simultaneous postdelivery diagnosis of TB in a mother and infant pair. PATIENT CONCERNS: A 28-year-old woman presented with a sudden onset of convulsions, loss of consciousness, coughing, fever, and breathing difficulty. Her 18-day-old infant daughter developed cough and wheezing. DIAGNOSIS: The mother's chest computed tomography showed diffuse interstitial changes and both lungs' exudation. Enhanced cranial magnetic resonance imaging showed scattered nodular intracranial lesions. A tuberculin skin test and an interferon-gamma release assay were negative. Xpert MTB/RIF (Xpert) testing and acid-fast bacilli smear of bronchoalveolar lavage (BAL) fluid of the mother were negative. Loop-mediated isothermal amplification of BAL fluid was positive for Mycobacterium tuberculosis, and next-generation sequencing confirmed the diagnosis of TB. A biopsy specimen also showed characteristic TB findings. The mother was diagnosed with TB and TB encephalitis. The infant's BAL fluid was positive for acid-fast bacilli and Xpert and, therefore, was diagnosed with TB. INTERVENTIONS: The mother was treated with rifampicin and isoniazid for 9 months, ethambutol and pyrazinamide for 3 months, and prednisolone acetate for 8 weeks. The infant received ventilator-assisted ventilation for 10 days and anti-tuberculous therapy for 11 months. OUTCOMES: After anti-tuberculous therapy, the mother and infant both gradually recovered. The mother's chest computed tomography showed significant recovery 9 months after discharge. The infant developed normally during the 11-month follow-up. LESSONS: This mother-child case pair highlights the value of loop-mediated isothermal amplification and next-generation sequencing as new diagnostic technologies for diagnosing TB in patients with multiple negative tests.

Incremental yield of Xpert® MTB/RIF Ultra over Xpert® MTB/RIF in the diagnosis of extrapulmonary TB.

BACKGROUND: Extrapulmonary TB (EPTB) comprises approximately 15-20% of TB cases worldwide, and its diagnosis is difficult. The sensitivity of Xpert® MTB/RIF (Xpert) in the diagnosis of EPTB is low on account of its paucibacillary nature. Xpert® MTB/RIF Ultra (Ultra) was developed to improve sensitivity.OBJECTIVE: To compare the sensitivity of Ultra test with that of Xpert against MGIT™ (Mycobacteria Growth Indicator Tube) culture and a composite reference standard (CRS).METHODS: We recruited consecutive treatment-naïve patients with suspected EPTB. Demographic information, clinical and relevant laboratory data were collected.RESULTS: From January 2019 to November 2019, 210 patients provided 250 samples. Against MGIT culture, the sensitivity of Ultra was significantly higher than Xpert (72% vs. 51.1%; P = 0.04), the specificity was lower (87.8% vs. 95.1%). Against the CRS also, the sensitivity of Ultra was significantly higher than Xpert (45.4% vs. 25.2%; P = 0.002); however, the specificities were similar (98.2% vs. 99.1%). The trend towards higher sensitivity of Ultra compared to Xpert was seen in most of the individual samples. The sensitivities against MGIT and CRS were as follows: lymph node (68.1% vs. 31.8%; P = 0.01) and (59.5% vs. 23.8%; P = 0.001), pleural biopsy (80.0% on both; P = NS) and (53.8% vs. 46.2%; P = NS) and pleural fluid (66.7% vs. 50%; P = NS) and (22.5% vs. 9.6%; P = NS), respectively.CONCLUSIONS: Xpert Ultra showed a significantly higher sensitivity in diagnosing EPTB than Xpert.

A Difficult Case of Pyrexia of Unknown Origin.

This is the case of a 15-y-old boy who presented with fever and back pain with MRI features of spondylitis. A CT-guided vertebral biopsy showed acute and chronic inflammatory cells and grew Pseudomonas aeruginosa on aerobic culture. The child was treated for 2 wk with antibiotics with no response. Meanwhile, he developed new lung, liver, and splenic lesions on CT imaging. Empiric antitubercular therapy was then started and continued for 8 wk during which time there was progressive clinical deterioration. At this time the patient underwent bronchoscopy with lavage and endoscopic ultrasound-guided subcarinal lymph node and lung biopsy. The Xpert MTB/Rif ULTRA was "trace positive" in the bronchoscopic lavage with indeterminate rifampicin resistance, while it was negative in lymph node and lung biopsy. The lymph node and lung biopsy histopathology showed nonspecific inflammatory changes with no granulomas or malignant cells. In view of the positive Xpert ULTRA with indeterminate rifampicin resistance and no response to first-line drugs, treatment with second-line antitubercular drugs was initiated. The clinical condition continued to deteriorate; here the imaging findings were reviewed again and repeat aspiration cytology and biopsy from intra-abdominal nodes was carried out. This yielded the diagnosis of Hodgkin lymphoma. The patient had stage IVB disease. He responded well to standard chemotherapy and is currently in remission. The case illustrates the need to avoid empiric therapy, repeat invasive procedures if so needed, choose the site/method of biopsy appropriately and interpret results of investigations carefully when evaluating a patient with pyrexia of unknown origin.

Xpert MTB/RIF Ultra Is Highly Sensitive for the Diagnosis of Tuberculosis Lymphadenitis in a High-HIV Setting.

Tuberculosis lymphadenitis (TBL) is the most common extrapulmonary tuberculosis (EPTB) manifestation. Xpert MTB/RIF Ultra (Ultra) is a World Health Organization-endorsed diagnostic test, but performance data for TBL, including on noninvasive specimens, are limited. Fine-needle aspiration biopsy specimens (FNABs) from outpatients (≥18 years) with presumptive TBL (n = 135) underwent (i) routine Xpert MTB/RIF testing (later with Ultra once programmatically available), (ii) MGIT 960 culture (if Xpert or Ultra negative or rifampicin resistant), and (iii) study Ultra testing. Concentrated paired urine specimens underwent Ultra testing. Primary analyses used a microbiological reference standard (MRS). In a head-to-head comparison (n = 92) of an FNAB study Ultra and Xpert, Ultra had increased sensitivity (91% [95% confidence interval: 79, 98] versus 72% [57, 84]; P = 0.016) and decreased specificity (76% [61, 87] versus 93% [82, 99]; P = 0.020) and diagnosed patients not on treatment. Neither HIV nor alternative reference standards affected sensitivity and specificity. In patients with both routine and study Ultra tests, the latter detected more cases (+20% [0, 42]; P = 0.034), and false-negative study Ultra results were more inhibited than true-positive results. Study Ultra false positives had less mycobacterial DNA than true positives (trace-positive proportions, 59% [13/22] versus 12% [5/51]; P < 0.001). "Trace" exclusion or recategorization removed potential benefits offered over Xpert. Urine Ultra tests had low sensitivity (18% [7, 35]). Ultra testing on FNABs is highly sensitive and detects more TBL than Xpert (Ultra still missed some cases due in part to inhibition). Patients with FNAB Ultra-positive "trace" results, most of whom will be culture negative, may require additional clinical investigation. Urine Ultra testing could reduce the number of patients needing invasive sampling.

Bioenergetic Heterogeneity in Mycobacterium tuberculosis Residing in Different Subcellular Niches.

ATP/ADP depicts the bioenergetic state of Mycobacterium tuberculosis (Mtb). However, the metabolic state of Mtb during infection remains poorly defined due to the absence of appropriate tools. Perceval HR (PHR) was recently developed to measure intracellular ATP/ADP levels, but it cannot be employed in mycobacterial cells due to mycobacterial autofluorescence. Here, we reengineered the ATP/ADP sensor Perceval HR into PHR-mCherry to analyze ATP/ADP in fast- and slow-growing mycobacteria. ATP/ADP reporter strains were generated through the expression of PHR-mCherry. Using the Mtb reporter strain, we analyzed the changes in ATP/ADP levels in response to antimycobacterial agents. As expected, bedaquiline induced a decrease in ATP/ADP. Interestingly, the transcriptional inhibitor rifampicin led to the depletion of ATP/ADP levels, while the cell wall synthesis inhibitor isoniazid did not affect the ATP/ADP levels in Mtb. The usage of this probe revealed that Mtb faces depletion of ATP/ADP levels upon phagocytosis. Furthermore, we observed that the activation of macrophages with interferon gamma and lipopolysaccharides leads to metabolic stress in intracellular Mtb. Examination of the bioenergetics of mycobacteria residing in subvacuolar compartments of macrophages revealed that the bacilli residing in phagolysosomes and autophagosomes have significantly less ATP/ADP than the bacilli residing in phagosomes. These observations indicate that phagosomes represent a niche for metabolically active Mtb, while autophagosomes and phagolysosomes harbor metabolically quiescent bacilli. Interestingly, even in activated macrophages, Mtb residing in phagosomes remains metabolically active. We further observed that macrophage activation affects the metabolic state of intracellular Mtb through the trafficking of Mtb from phagosomes to autophagosomes and phagolysosomes. IMPORTANCE ATP/ADP levels guide bacterial cells, whether to replicate or to enter nonreplicating persistence. However, tools for measuring ATP/ADP levels with spatiotemporal resolution are lacking. Here, we describe a method for tracking ATP/ADP levels at the single-cell and population levels. Using this tool, we have demonstrated that the transcription inhibitor rifampicin induces metabolic stress. In contrast, the cell wall synthesis inhibitor isoniazid does not alter the metabolic state of the bacilli, suggesting that transcription is tightly intertwined with metabolism, while cell wall synthesis is not. Furthermore, we analyzed the metabolic state of mycobacteria residing in different compartments of macrophages. We observed that Mtb cells residing inside phagosomes have healthy ATP/ADP levels. In contrast, the bacteria residing inside phagolysosomes and autophagosomes face depletion of ATP. Interestingly, the activation of macrophages facilitates the trafficking of mycobacterial cells from metabolism-conducive phagosomes to metabolism-averse phagolysosomes and autophagosomes. We believe that this tool holds the key to the identification of inhibitors of mycobacterial metabolism.

Pharmacokinetics of oral antimycobacterials and dosing guidance for Mycobacterium avium complex treatment in cystic fibrosis.

BACKGROUND: Treatment failure of Mycobacterium avium complex (MAC) pulmonary disease occurs in about 30% of people with cystic fibrosis (CF) and may be a result of abnormal drug concentrations. METHODS: Prospective, cross-over, single-dose PK study of 20 pancreatic insufficient individuals with CF and 10 healthy controls (HC). CF subjects received simultaneous doses of oral azithromycin, ethambutol, and rifampin in the fasting state and with food and pancreatic enzymes, separated by two weeks. HC received fasting doses only. A non-compartmental model was used to estimate PK parameters of drugs and metabolites. RESULTS: Azithromycin maximum concentration (Cmax ) was higher and rifampin Cmax was lower in fasting CF subjects compared to HC, while other PK measures, including those for ethambutol, were similar. Addition of food and enzymes did not improve the Cmax of the antimycobacterial drugs. Nineteen of 20 CF subjects had one or more abnormal Cmax z-scores in either the fasting or fed state (or both), when compared to HC. CONCLUSION: PK profiles of azithromycin and ethambutol were similar between CF and HC, except azithromycin Cmax was slightly higher in people with CF after a single dose. Rifampin PK parameters were altered in persons with CF. Addition of food and enzymes in CF subjects did not improve PK parameters. Standard dosing guidelines should be used as a starting point for people with CF initiating MAC therapy and therapeutic drug monitoring should be routinely performed to prevent the possibility of treatment failure due to abnormal drug concentrations. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02372383 Prior abstract publication: 1. Martiniano S, Wagner B, Brennan L, Wempe M, Anderson P, Nick J, Sagel S. Pharmacokinetics of oral MAC antibiotics in cystic fibrosis. Am J Resp Crit Care Med A4842-A4842, 2017. 2. Martiniano SL, Wagner BD, Brennan L, Wempe MF, Anderson PL, Nick JA, Sagel SD. Pharmacokinetics of oral MAC antibiotics in cystic fibrosis. J Cyst Fibros 16: S52-53, 2017.

Tale of compounding oddities.

We present a case of a 59-year-old man, who on being evaluated for abdominal pain and headache, was found to have a pancreatic head mass and inflammatory hypophysitis. Xpert MTB/Rif of the pancreatic mass biopsy showed the presence of tuberculosis (TB) with a very low load, and rifampicin resistance was detected with absence of probes A and B. Pyrosequencing (a novel genotypic test for TB) of the Xpert MTB/Rif isolate detected a single, rare, high-confidence mutation (S512T) in the rpoB region (rifampicin resistance determining region in the MTB genome). The TB mycobacteria growth indicator tube (TBMGIT) phenotypic drug susceptibility test (DST), however, showed rifampicin susceptibility. Incidentally, he was unable to tolerate rifampicin and responded well to a non-rifampicin-based regimen. We discuss a possible hypothesis of the Xpert-DST discordance in accordance with a recent literature review on phenotypic DST methods. We also discuss the utility of pyrosequencing in clinical practice for the diagnosis of TB and its resistance patterns.

Central nervous system tuberculosis.

PURPOSE OF REVIEW: Central nervous system (CNS) tuberculosis is the most devastating form of tuberculosis (TB), with mortality and or neurological sequelae in over half of individuals. We reviewed original research and systematic reviews published since 1 January 2019 for new developments in CNS TB pathophysiology, diagnosis, management and prognosis. RECENT FINDINGS: Insight in the pathophysiology is increasing steadily since the landmark studies in 1933, focussing on granuloma type classification, the relevance of the M. tuberculosis bacterial burden and the wide range of immunological responses. Although Xpert/RIF has been recommended by the WHO for extrapulmonary TB diagnosis, culture is still needed to increase the sensitivity of TB meningitis diagnosis. Sequential MRIs can improve understanding of neurological deficits at baseline and during treatment. Pharmacokinetic/pharmacodynamic modelling suggests that higher doses of rifampicin and isoniazid in TB meningitis could improve survival. SUMMARY: Recent studies in the field of CNS-TB have largely focussed on TB meningitis. The outcome may improve by optimizing treatment dosing. This needs to be confirmed in clinical trials. Due to the important role of inflammation, these trials should be used as the platform to study the inflammatory and metabolomic responses. This could improve understanding of the biology of this disease and improve patient outlook by enabling individualised host-directed therapy.

Tuberculosis con resistencia a rifampicina en Chile / Tuberculosis with resistance to rifampicin in Chile

Resumen El éxito de los tratamientos acortados de la tuberculosis se debe a la asociación de fármacos bactericidas y esterilizantes, principalmente Rifampicina e Isoniazida. Cuando la Rifampicina no puede ser utilizada por resistencia, los tratamientos son más prolongados y el éxito en la curación se reduce. La resistencia a Rifampicina frecuentemente se acompaña de resistencia a Isoniazida (Multidrogoresistencia o MDR). La OMS informa que en 2019 se diagnosticaron sólo el 44% de los casos estimados de tuberculosis con resistencia a Rifampicina (se proyectaba 465.000 casos) y se trató sólo al 38% de los casos estimados, quedando una gran proporción de casos sin diagnosticar y sin tratar. En Chile la vigilancia de la susceptibilidad a fármacos de primera línea en cepas de Mycobacterium tuberculosis se efectúa mediante biología molecular desde 2014, observándose un progresivo incremento de casos resistentes a Rifampicina desde 1% (23 casos) para ese año hasta 2,2% (65 casos) en 2019. La mayoría de casos de resistencia a Rifampicina corresponden a resistencia inicial. En casos con resistencia a Rifampicina realizamos estudio de susceptibilidad a fármacos de segunda línea en el laboratorio de referencia nacional. La terapia de TB-MDR tradicional tiene baja eficacia, con abandonos frecuentes por su largo tiempo de terapia y toxicidad. Nuevos tratamientos sin inyectables y el uso de Clofazimina, Fluorquinolonas, Linezolid y Bedaquilina tienen una mejor tasa de curación. Recientemente, el Programa de Control de la Tuberculosis de Chile dispone de esta terapia más eficaz y de menor duración por vía oral con estos fármacos.

The high success rate of shortened Tuberculosis (TB) treatments has been achieved by the association of bactericidal and sterilizing drugs. The main drugs are Rifampicin and Isoniazide. When Rifampicin cannot be used by resistance, the treatment is prolonged and success in healing is significantly reduced. Resistance to Rifampicin is often accompanied by resistance to Isoniazide (Multidrug resistance or MDR). WHO reports that only 44% of estimated TB cases with resistance to Rifampicin were diagnosed in 2019 (465,000 cases projected) and only 38% of estimated cases were treated, with a large proportion of cases remaining undiagnosed and untreated. In Chile, monitoring of susceptibility to first-line drugs is conducted in strains of Mycobacterium tuberculosis by molecular biology since 2014, observing a progressive increase in cases with Rifampicin resistance from 1% for that year (23 cases) to 2.2% in 2019 (65 cases). Most cases of resistance to Rifampicin correspond to cases of initial resistance. In cases with resistance to Rifampicin we carry out susceptibility study to second-line drugs in a national reference laboratory. MDR-TB therapy has low efficacy, with frequent abandonments for its long therapy time and toxicity. New non-injectable treatments and use of Clofazimine, Fluorquinolones, Linezolid and Bedaquiline are achieving a better cure rate. Recently, Chile's TB Control Program has this most effective and shorter-lasting oral therapy with these drugs.