Xpert MTB/XDR: a 10-Color Reflex Assay Suitable for Point-of-Care Settings To Detect Isoniazid, Fluoroquinolone, and Second-Line-Injectable-Drug Resistance Directly from Mycobacterium tuberculosis-Positive Sputum.

We describe the design, development, analytical performance, and a limited clinical evaluation of the 10-color Xpert MTB/XDR assay (CE-IVD only, not for sale in the United States). This assay is intended as a reflex test to detect resistance to isoniazid (INH), fluoroquinolones (FLQ), ethionamide (ETH), and second-line injectable drugs (SLIDs) in unprocessed sputum samples and concentrated sputum sediments which are positive for Mycobacterium tuberculosis The Xpert MTB/XDR assay simultaneously amplifies eight genes and promoter regions in M. tuberculosis and analyzes melting temperatures (Tm s) using sloppy molecular beacon (SMB) probes to identify mutations associated with INH, FLQ, ETH, and SLID resistance. Results can be obtained in under 90 min using 10-color GeneXpert modules. The assay can differentiate low- versus high-level resistance to INH and FLQ as well as cross-resistance versus individual resistance to SLIDs by identifying mutation-specific Tm s or Tm patterns generated by the SMB probes. The assay has a limit of detection comparable to that of the Xpert MTB/RIF assay and successfully detected 16 clinically significant mutations in a challenge set of clinical isolate DNA. In a clinical study performed at two sites with 100 sputum and 214 clinical isolates, the assay showed a sensitivity of 94% to 100% and a specificity of 100% for all drugs except for ETH compared to that of sequencing. The sensitivity and specificity were in the same ranges as those of phenotypic drug-susceptibility testing. Used in combination with a primary tuberculosis diagnostic test, this assay should expand the capacity for detection of drug-resistant tuberculosis near the point of care.

Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV Test.

With the approach of respiratory virus season in the Northern Hemisphere, clinical microbiology and public health laboratories will need rapid diagnostic assays to distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza virus and respiratory syncytial virus (RSV) infections for diagnosis and surveillance. In this study, the clinical performance of the Xpert Xpress SARS-CoV-2/Flu/RSV test (Cepheid, Sunnyvale, CA, USA) for nasopharyngeal swab specimens was evaluated in four centers: Johns Hopkins Medical Microbiology Laboratory, Northwell Health Laboratories, NYC Public Health Laboratory, and Los Angeles County/University of Southern California (LAC+USC) Medical Center. A total of 319 nasopharyngeal swab specimens, positive for SARS-CoV-2 (n = 75), influenza A virus (n = 65), influenza B virus (n = 50), or RSV (n = 38) or negative (n = 91) by the standard-of-care nucleic acid amplification tests at each site, were tested using the Cepheid panel test. The overall positive percent agreement for the SARS-CoV-2 target was 98.7% (n = 74/75), and the negative agreement was 100% (n = 91), with all other analytes showing 100% total agreement (n = 153). Standard-of-care tests to which the Cepheid panel was compared included the Cepheid Xpert Xpress SARS-CoV-2, Cepheid Xpert Xpress Flu/RSV, GenMark ePlex respiratory panel, BioFire respiratory panel 2.1 and v1.7, DiaSorin Simplexa COVID-19 Direct, and Hologic Panther Fusion SARS-CoV-2 assays. The Xpert Xpress SARS-CoV-2/Flu/RSV test showed high sensitivity and accuracy for all analytes included in the test. This test will provide a valuable clinical diagnostic and public health solution for detecting and differentiating SARS-CoV-2, influenza A and B virus, and RSV infections during the current respiratory virus season.

Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test.

Nucleic acid amplification tests (NAATs) are the primary means of identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and isolation resources and allow rapid therapeutic interventions. We evaluated the analytical and clinical performance characteristics of the Xpert Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2; other infectious coronavirus species, including SARS-CoV; and 85 nasopharyngeal swab specimens positive for other respiratory viruses, including endemic human coronaviruses (hCoVs). Clinical performance was assessed using 483 remnant upper- and lower-respiratory-tract specimens previously analyzed by standard-of-care (SOC) NAATs. The limit of detection of the Xpert test was 0.01 PFU/ml. Other hCoVs, including Middle East respiratory syndrome coronavirus, were not detected by the Xpert test. SARS-CoV, a closely related species in the subgenus Sarbecovirus, was detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 219/220 (99.5%), and the negative agreement was 250/261 (95.8%). A third tie-breaker NAAT resolved all but three of the discordant results in favor the Xpert test. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a variety of upper- and lower-respiratory-tract specimens. The high sensitivity and short time to results of approximately 45 min may impact patient management.

Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System.

Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.

Automatic Identification of Individual rpoB Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay.

Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the rpoB gene by the use of melting temperature (Tm ) information from 4 rpoB probes which can fall in one of the 9 different assay-specified Tm windows. The large amount of Tm data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the rpoB RRDR by utilizing the pattern of the Tm of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific "Tm signature" reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the Tm distances between the test sample and the reference Tm values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of rpoB single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.

Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System.

Bacillus anthracis is a tier 1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect B. anthracis bacteremia using a system that is suitable for point-of-care testing. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. The assay limit of detection (LOD) and dynamic range were determined by spiking B. anthracis DNA into individual PCR mixtures and B. anthracis CFU into human blood. One-milliliter blood samples were added to the filter-based detection cartridge and tested for B. anthracis on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates or by testing blood samples drawn from patients with concurrent non-B. anthracis bacteremia or nonbacteremic controls. The assay LODs were 5 genome equivalents per reaction and 10 CFU/ml blood for both the B. anthracis Sterne and V1B strains. There was a 6-log10 dynamic range. Assay specificity was 100% for tests of non-B. anthracis bacterial isolates and patient blood samples. Assay time was less than 90 min. This automated system suitable for point-of-care detection rapidly identifies B. anthracis directly from blood with high sensitivity. This assay might lead to early detection and more rapid therapy in the event of a bioterrorism attack.

Enhancing Patient Selection in Stage IIIA-IIIB NSCLC: Invasive Lymph Node Restaging after Neoadjuvant Therapy.

Restaging of mediastinal lymph nodes plays a crucial role in the multimodal treatment of stage IIIA Non-Small-Cell Lung Cancer (NSCLC). This study aimed to assess the impact of restaging using endobronchial ultrasound (EBUS), endoesophageal ultrasound (EUS), and transcervical extended mediastinal lymphadenectomy (TEMLA) after neoadjuvant chemotherapy (CHT) or chemoradiotherapy (CRT) on the 5-year overall survival (OS) of patients with NSCLC diagnosed with clinical stage IIIA-IIIB and metastatic ipsilateral mediastinal nodes (N2) who underwent radical pulmonary resections. Patients diagnosed with stage IIIA-IIIB NSCLC and N2 mediastinal nodes were included in this study. Restaging of mediastinal lymph nodes was performed using EBUS, EUS, and TEMLA. The patients were divided into two groups based on the restaging method: the TEMLA restaging group and the chest CT scan-only group. The primary outcome measure was the 5-year OS rate, and the secondary outcome measures included median OS and survival percentages. Statistical analysis, including the log-rank test, was conducted to assess the differences between the two groups. The TEMLA restaging group demonstrated significantly better overall survival compared to the chest CT scan-only group (log-rank test, p = 0.02). This was evident through a four-fold increase in median OS (59 vs. 14 months) and a higher 5-year OS rate of 55.9% (95% CI: 40.6-71.1) compared to 25.0% (95% CI: 13.7-36.3) in the chest CT scan-only group (p = 0.003). Invasive restaging of mediastinal lymph nodes improves the selection of patients with stage IIIA-IIIB (N2) NSCLC after neoadjuvant therapy. The use of EBUS, EUS, and TEMLA provides valuable information for identifying patients who may benefit from surgery by identifying N2 to N0-1 downstaging. These findings emphasize the importance of incorporating restaging procedures into the treatment decision-making process for NSCLC patients with mediastinal lymph node involvement.

Merkel cell carcinoma - description of five cases.

Merkel cell carcinoma (MCC) is a rare but very aggressive skin cancer that derives from neuroendocrine cells of the skin. Merkel cell carcinoma morbidity has been continuously increasing for the last few years. Increasing reported incidence of MCC is probably connected with increasing occurrence of this kind of malignancy or with development of histological and immunohistochemical methods of sample examinations which have allowed for more precise diagnosis of skin tumor that might have previously not been accurately recognized. Merkel cell carcinoma develops as nodules early recognized as basocellular carcinoma, squamous cell carcinoma, amelanotic melanoma or skin lymphoma. Merkel cell carcinoma can be morphologically similar to skin metastasis as well as mild changes such as lipoma, cysts, fibroma or vessel changes. Accurate diagnosis is very important because it determines successful management and risk of progression of disease. We describe 5 patients with MCC who underwent surgical excision and then, after estimation of stage of disease, complementary treatment. Our observations prove that every tumor with MCC should be cut out with wide margins and regional lymphadenectomy or sentinel node biopsy is compulsory. After cutting out MCC involved-field radiotherapy is necessary and improves prognosis. Presence of metastases in lymphatic nodes is an indication for complementary chemotherapy.

Assessment of connective tissue metabolism in patients with head and neck malignancies treated with radiotherapy.

<br><b>Introduction:</b> Despite the use of highly specialized irradiation techniques in the treatment of head and neck tumors, it is still impossible to selectively destroy cancer cells without damaging normal structures, including connective tissue cells.</br> <br><b>Aim:</b> The aim of the study was to analyze the concentration of degradation markers such as collagen type I (carboxyterminal telopeptide of type I collagen; ICTP) and elastin (elastin-derived peptides; EDPs) as well as selected metalloproteinases (MMP-1, MMP-2, MMP-9) in patients with head and neck malignancies undergoing radiotherapy.</br> <br><b>Material and methods:</b> The test group consisted of 56 men, who underwent radical or palliative radiotherapy. The concentrations of ICTP, EDPs, MMP-1, MMP-2, MMP-9 were determined in three blood samples collected from patients prior to radiotherapy, immediately after its completion and 3 months after the therapy.</br> <br><b>Results</b>: Both radical and palliative radiotherapy contribute to a significant increase in the concentration of EDPs. At the time of healing of post-irradiation lesions, the level of EDPs was reduced in both groups. The ICTP concentration was not affected by radiotherapy. No significant differences were observed in the concentration of MMP-1 and MMP-2 before and after radiotherapy. Radical radiotherapy caused a statistically significant late reduction in the concentration of MMP-9. The lowest concentrations of MMP-1, MMP-2, MMP-9 in the serum of patients qualified for palliative radiotherapy were recorded in a samples collected three months post-irradiation.</br> <br><b>Conclusions:</b> The degradation markers of key extracellular matrix structural proteins may be helpful tools in the objective assessment of radiation-induced injuries to the connective tissue.</br>.

Atypical dissemination of lung cancer to the adrenal gland and to the spleen.

Metastases of lung cancer to such organs as the liver, bones or to the central nervous system appear to be a frequent complication of this disease. At the same time, metastases to the adrenal gland are found less frequently. Metastases of lung cancer to the spleen are a great rarity and they are described sporadically. Our report presents a unique case of left lung cancer with simultaneous metastases to the adrenal gland and to the spleen. All the presented lesions were diagnosed by ultrasound guided biopsy and confirmed by histopathological examination. The patient received combined chemoradiotherapy. She was closely monitored over an 18-month observation period following treatment. No new metastases were reported.

Fully body visual self-modeling of robot morphologies.

Internal computational models of physical bodies are fundamental to the ability of robots and animals alike to plan and control their actions. These "self-models" allow robots to consider outcomes of multiple possible future actions without trying them out in physical reality. Recent progress in fully data-driven self-modeling has enabled machines to learn their own forward kinematics directly from task-agnostic interaction data. However, forward kinematic models can only predict limited aspects of the morphology, such as the position of end effectors or velocity of joints and masses. A key challenge is to model the entire morphology and kinematics without prior knowledge of what aspects of the morphology will be relevant to future tasks. Here, we propose that instead of directly modeling forward kinematics, a more useful form of self-modeling is one that could answer space occupancy queries, conditioned on the robot's state. Such query-driven self-models are continuous in the spatial domain, memory efficient, fully differentiable, and kinematic aware and can be used across a broader range of tasks. In physical experiments, we demonstrate how a visual self-model is accurate to about 1% of the workspace, enabling the robot to perform various motion planning and control tasks. Visual self-modeling can also allow the robot to detect, localize, and recover from real-world damage, leading to improved machine resiliency.

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

BACKGROUND: Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. METHODS: We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. RESULTS: SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. CONCLUSION: eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

Sample collection and transport strategies to enhance yield, accessibility, and biosafety of COVID-19 RT-PCR testing.

Introduction. Non-invasive sample collection and viral sterilizing buffers have independently enabled workflows for more widespread COVID-19 testing by reverse-transcriptase polymerase chain reaction (RT-PCR).Gap statement. The combined use of sterilizing buffers across non-invasive sample types to optimize sensitive, accessible, and biosafe sampling methods has not been directly and systematically compared.Aim. We aimed to evaluate diagnostic yield across different non-invasive samples with standard viral transport media (VTM) versus a sterilizing buffer eNAT- (Copan diagnostics Murrieta, CA) in a point-of-care diagnostic assay system.Methods. We prospectively collected 84 sets of nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT-PCR-confirmed patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal swabs, oral swabs, and saliva were placed in either VTM or eNAT, prior to testing with the Xpert Xpress SARS-CoV-2 (Xpert). The sensitivity of each sampling strategy was compared using a composite positive standard.Results. Swab specimens collected in eNAT showed an overall superior sensitivity compared to swabs in VTM (70 % vs 57 %, P=0.0022). Direct saliva 90.5 %, (95 % CI: 82 %, 95 %), followed by NP swabs in VTM and saliva in eNAT, was significantly more sensitive than nasal swabs in VTM (50 %, P<0.001) or eNAT (67.8 %, P=0.0012) and oral swabs in VTM (50 %, P<0.0001) or eNAT (58 %, P<0.0001). Saliva and use of eNAT buffer each increased detection of SARS-CoV-2 with the Xpert; however, no single sample matrix identified all positive cases.Conclusion. Saliva and eNAT sterilizing buffer can enhance safe and sensitive detection of COVID-19 using point-of-care GeneXpert instruments.

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

BACKGROUND: Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. METHODS: We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream RT-PCR testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. RESULTS: SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log 10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on vero-E6 cell lines was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. CONCLUSION: eNAT and similar guanidinium thiocyanate-based media may be of value for transport, preservation, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

Evaluation of sample collection and transport strategies to enhance yield, accessibility, and biosafety of COVID-19 RT-PCR testing.

Sensitive, accessible, and biosafe sampling methods for COVID-19 reverse-transcriptase polymerase chain reaction (RT-PCR) assays are needed for frequent and widespread testing. We systematically evaluated diagnostic yield across different sample collection and transport workflows, including the incorporation of a viral inactivation buffer. We prospectively collected nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT-PCR-confirmed patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal and oral swabs were placed in both viral transport media (VTM) and eNAT™, a sterilizing transport buffer, prior to testing with the Xpert Xpress SARS-CoV-2 (Xpert) test. The sensitivity of each sampling strategy was compared using a composite positive standard. Overall, swab specimens collected in eNAT showed superior sensitivity compared to swabs in VTM (70% vs 57%, P=0.0022). Direct saliva 90.5%, (95% CI: 82%, 95%), followed by NP swabs in VTM and saliva in eNAT, was significantly more sensitive than nasal swabs in VTM (50%, P<0.001) or eNAT (67.8%, P=0.0012) and oral swabs in VTM (50%, P<0.0001) or eNAT (56%, P<0.0001). Saliva and use of eNAT buffer each increased detection of SARS-CoV-2 with the Xpert test; however, no single sample matrix identified all positive cases.

Task-agnostic self-modeling machines.

A robot modeled itself without prior knowledge of physics or its shape and used the self-model to perform tasks and detect self-damage.

Autonomous drone hunter operating by deep learning and all-onboard computations in GPS-denied environments.

This paper proposes a UAV platform that autonomously detects, hunts, and takes down other small UAVs in GPS-denied environments. The platform detects, tracks, and follows another drone within its sensor range using a pre-trained machine learning model. We collect and generate a 58,647-image dataset and use it to train a Tiny YOLO detection algorithm. This algorithm combined with a simple visual-servoing approach was validated on a physical platform. Our platform was able to successfully track and follow a target drone at an estimated speed of 1.5 m/s. Performance was limited by the detection algorithm's 77% accuracy in cluttered environments and the frame rate of eight frames per second along with the field of view of the camera.

Collagen Type III Metabolism Evaluation in Patients with Malignant Head and Neck Cancer Treated with Radiotherapy.

Ionizing radiation affects the metabolism of key proteins of extracellular matrix including type III collagen, an important component of human skin. The aim of the work is an analysis of the impact of radical and palliative radiotherapy on collagen type III synthesis in patients with head and neck cancer. The test group consisted of 56 males with histopathologically confirmed head and neck cancer, for whom radiotherapy was applied as a form of radical or palliative treatment. The level of procollagen III aminoterminal propeptide (PIIINP), which is a marker of collagen type III synthesis, was determined in blood serum before radiotherapy, immediately following radiotherapy, and 3 months after it was finished. As a result of radical radiotherapy a statistically significant decrease of PIIINP levels in serum (p < 0.0001) was observed, both immediately after the radiotherapy and 3 months after the end of the treatment. Also the palliative radiotherapy caused a significant decrease of PIIINP right after the treatment (p = 0.0052), as well as during the examination performed 3 months later (p = 0.0004). The achieved results suggest that PIIINP can be used as a marker helpful in assessing radiation damage to connective tissue.

Non-intercostal access for video-assisted thoracic surgery-analysis of technical advantages and disadvantages.

BACKGROUND: Video-assisted thoracoscopic surgery (VATS) lobectomy has become an accepted method for the treatment of early-stage non-small-cell lung cancer (NSCLC). The standard VATS approach is an intercostal one which is often followed by postoperative pain due to injury of the intercostal nerve. The non-intercostal techniques of VATS include the subxiphoid, transcervical, transdiaphragmatic and transoral procedures. METHODS: The technical difficulty of operative management of the anatomical structures during VATS anatomical resection are compared for the intercostal, subxiphoid and transcervical approaches. RESULTS: Some operative steps have different range of difficulty, which are analyzed in detail. CONCLUSIONS: The clearest advantages of the non-intercostal approaches include less postoperative pain and superradial bilateral mediastinal lymphadenectomy in case of the transcervical approach. However, the non-intercostal approaches are more technically demanding procedures, which therapeutic role has to be clarified in the future.

The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing.

The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection.IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R.