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Although tuberculosis is slowly decreasing, nontuberculous mycobacterial lung disease is significantly increasing. We describe new methods and applications for faster turnaround times in the diagnosis of tuberculosis and nontuberculous mycobacterial lung disease and have included the latest mycobacterial taxonomy. Although the focus is mainly on molecular assays, we also discuss improvements of acid-fast bacilli smear microscopy and stress the need for performing minimal inhibitory concentration determinations especially for tuberculosis. Additionally, important considerations for negative nucleic acid amplification assay results used for releasing tuberculosis suspects from airborne infection isolation rooms saving precious resources for the health care system, are also included.

In 2019, tuberculosis is still a global source of morbidity and mortality. To determine and provide the most effective treatment regimen to patients, the tuberculosis laboratory needs to rapidly but reliably answer 2 main questions: (1) Is Mycobacterium tuberculosis detectable in the patient specimen? and (2) If so, is the strain detected drug susceptible or does it show any form of drug resistance? In cases of drug resistance, health care providers need to have access to minimal inhibitory concentration results and to the type of mutation conferring drug resistance to tailor the most appropriate drug regimen.

Additional file 1. Materials provided in the online additional file include: (1) Engineering design of medium pressure reservoir (MPR); (2) Figure S1, Detailed schematic of MPR; (3) Table S1, Description of components of MPR; and (4) Figure S2, Pilot clinical trial profile.

Detection of tuberculosis at the point-of-care (POC) is limited by the low sensitivity of current commercially available tests. We describe a diagnostic accuracy field evaluation of a prototype urine Tuberculosis Lipoarabinomannan Lateral Flow Assay (TB-LAM LFA) in both HIV-positive and HIV-negative patients using fresh samples with sensitivity and specificity as the measures of accuracy. This prototype combines a proprietary concentration system with a sensitive LFA. In a prospective study of 292 patients with suspected pulmonary tuberculosis in Uganda, the clinical sensitivity and specificity was compared against a microbiological reference standard including sputum Xpert MTB/RIF Ultra and solid and liquid culture. TB-LAM LFA had an overall sensitivity of 60% (95%CI 51–69%) and specificity of 80% (95%CI 73–85%). When comparing HIV-positive (N = 86) and HIV-negative (N = 206) patients, there was no significant difference in sensitivity (sensitivity difference 8%, 95%CI -11% to +24%, p = 0.4351) or specificity (specificity difference -9%, 95%CI -24% to +4%, p = 0.2051). Compared to the commercially available Alere Determine TB-LAM Ag test, the TB-LAM LFA prototype had improved sensitivity in both HIV-negative (difference 49%, 95%CI 37% to 59%, p200cells/μL (difference 59%, 95%CI 32% to 75%, p = 0.0009). This report is the first to show improved performance of a urine TB LAM test for HIV-negative patients in a high TB burden setting. We also offer potential assay refinement solutions that may further improve sensitivity and specificity.

BackgroundThe World Health Organization (WHO) has endorsed the next-generation Xpert MTB/RIF Ultra (Ultra) cartridge, and Uganda is currently transitioning from the older generation Xpert MTB/RIF (Xpert) cartridge to Ultra as the initial diagnostic test for pulmonary tuberculosis (TB). We assessed the diagnostic accuracy of Ultra for pulmonary TB among adults in Kampala, Uganda.MethodsWe sampled adults referred for Xpert testing at two hospitals and a health center over a 12-month period. We enrolled adults with positive Xpert and a random 1:1 sample with negative Xpert results. Expectorated sputum was collected for Ultra, and for solid and liquid culture testing for Xpert-negative patients. We measured sensitivity and specificity of Ultra overall and by HIV status, prior history of TB, and hospitalization, in reference to Xpert and culture results. We also assessed how classification of results in the new “trace” category affects Ultra accuracy.ResultsAmong 698 participants included, 211 (30%) were HIV-positive and 336 (48%) had TB. The sensitivity of Ultra was 90.5% (95% CI 86.8–93.4) and specificity was 98.1% (95% CI 96.1–99.2). There were no significant differences in sensitivity and specificity by HIV status, prior history of TB or hospitalization. Xpert and Ultra results were concordant in 670 (96%) participants, with Ultra having a small reduction in specificity (difference 1.9, 95% CI 0.2 to 3.6,p=0.01). When “trace” results were considered positive for all patients, sensitivity increased by 2.1% (95% CI 0.3 to 3.9,p=0.01) without a significant reduction in specificity (− 0.8, 95% CI − 0.3 to 2.0,p=0.08).ConclusionsAfter 1 year of implementation, Ultra had similar performance to Xpert. Considering “trace” results to be positive in all patients increased case detection without significant loss of specificity. Longitudinal studies are needed to compare the benefit of greater diagnoses to the cost of overtreatment.

Oral swab analysis (OSA) has been shown to detectMycobacterium tuberculosis(MTB) DNA in patients with pulmonary tuberculosis (TB). In previous analyses, qPCR testing of swab samples collected from tongue dorsa was up to 93% sensitive relative to sputum GeneXpert, when 2 swabs per patient were tested. The present study modified sample collection methods to increase sample biomass and characterized the viability of bacilli present in tongue swabs. A qPCR targeting conserved bacterial ribosomal rRNA gene (rDNA) sequences was used to quantify bacterial biomass in samples. There was no detectable reduction in total bacterial rDNA signal over the course of 10 rapidly repeated tongue samplings, indicating that swabs collect only a small portion of the biomass available for testing. Copan FLOQSwabs collected ~2-fold more biomass than Puritan PurFlock swabs, the best brand used previously (p = 0.006). FLOQSwabs were therefore evaluated in patients with possible TB in Uganda. A FLOQSwab was collected from each patient upon enrollment (Day 1) and, in a subset of sputum GeneXpert Ultra-positive patients, a second swab was collected on the following day (Day 2). Swabs were tested for MTB DNA by manual IS6110-targeted qPCR. Relative to sputum GeneXpert Ultra, single-swab sensitivity was 88% (44/50) on Day 1 and 94.4% (17/18) on Day 2. Specificity was 79.2% (42/53). Among an expanded sample of Ugandan patients, 62% (87/141) had colony-forming bacilli in their tongue dorsum swab samples. These findings will help guide further development of this promising TB screening method.

The cobas MTB and MTB-RIF/INH assays allow for detection of Mycobacterium tuberculosis complex (MTBC) nucleic acid and rifampicin (RIF) and isoniazid (INH) resistance-associated mutations in an automated, high-throughput workflow. In this study, we evaluated the performance of these assays, employing samples from settings of low and high tuberculosis (TB) burdens., The Roche cobas MTB and MTB-RIF/INH assays allow for detection of Mycobacterium tuberculosis complex (MTBC) nucleic acid and rifampicin (RIF) and isoniazid (INH) resistance-associated mutations in an automated, high-throughput workflow. In this study, we evaluated the performance of these assays, employing samples from settings of low and high tuberculosis (TB) burdens. A total of 325 frozen, leftover respiratory samples collected from treatment-naive patients with presumptive TB in Germany (n = 280) and presumptive RIF-resistant TB in Sierra Leone (n = 45) were used in this study. cobas MTB results for detection of MTBC DNA from N-acetyl-l-cysteine–sodium hydroxide (NALC–NaOH)-treated samples were compared to culture results. Predictions of RIF and INH resistance by the cobas MTB-RIF/INH assay were compared to a composite reference standard (phenotypic drug susceptibility testing and line probe assay). Whole-genome sequencing was used to resolve discordances. The overall sensitivity of cobas MTB for detection of MTBC DNA in culture-positive samples (n = 102) was 89.2% (95% confidence interval [CI], 81.7 to 93.9%). The specificity of cobas MTB was 98.6% (95% CI, 96.1 to 99.5%). Sensitivity and specificity for detection of RIF and INH resistance were 88.4% (95% CI, 75.5 to 94.9%) and 97.6% (95% CI, 87.4 to 99.6%) and 76.6% (95% CI, 62.8 to 86.4%) and 100.0% (95% CI, 90.8 to 100.0%), respectively. Discordant results for RIF and INH resistance were mainly due to uncommon mutations in samples from Sierra Leone that were not covered by the cobas MTB-RIF/INH assay. In conclusion, cobas MTB and MTB-RIF/INH assays provide accurate detection of MTBC DNA and resistance-associated mutations in respiratory samples. The influence of regional variations in the prevalence of resistance-conferring mutations requires further investigation.

Mycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing., Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis. We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

BACKGROUND: The increasing global prevalence of pulmonary nontuberculous mycobacteria (NTM) disease has called attention to challenges in NTM diagnosis and management. This study was conducted to understand management and outcomes of patients with pulmonary NTM disease at diverse centers across the United States. METHODS: We conducted a 10-year (2005–2015) retrospective study at 7 Vaccine and Treatment Evaluation Units to evaluate pulmonary NTM treatment outcomes in human immunodeficiency virus–negative adults. Demographic and clinical information was abstracted through medical record review. Microbiologic and clinical cure were evaluated using previously defined criteria. RESULTS: Of 297 patients diagnosed with pulmonary NTM, the most frequent NTM species were Mycobacterium avium-intracellulare complex (83.2%), M. kansasii (7.7%), and M. abscessus (3.4%). Two hundred forty-five (82.5%) patients received treatment, while 45 (15.2%) were followed without treatment. Eighty-six patients had available drug susceptibility results; of these, >40% exhibited resistance to rifampin, ethambutol, or amikacin. Of the 138 patients with adequate outcome data, 78 (56.5%) experienced clinical and/or microbiologic cure. Adherence to the American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) treatment guidelines was significantly more common in patients who were cured (odds ratio, 4.5, 95% confidence interval, 2.0–10.4; P

Additional file 1: Appendix 1. Supplementary Methods. Appendix 2. Tolerability questionnaire. Appendix 3. Supplemental figure.

Approximately 3.6 million cases of active tuberculosis (TB) go potentially undiagnosed annually, partly due to limited access to confirmatory diagnostic tests, such as molecular assays or mycobacterial culture, in community and primary healthcare settings. This article provides guidance for TB triage test evaluations. A TB triage test is designed for use in people with TB symptoms and/or significant risk factors for TB. Triage tests are simple and low-cost tests aiming to improve ease of access and implementation (compared with confirmatory tests) and decrease the proportion of patients requiring more expensive confirmatory testing. Evaluation of triage tests should occur in settings of intended use, such as community and primary healthcare centers. Important considerations for triage test evaluation include study design, population, sample type, test throughput, use of thresholds, reference standard (ideally culture), and specimen flow. The impact of a triage test will depend heavily on issues beyond accuracy, primarily centered on implementation.

Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood. Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis, Salmonella enterica, Aspergillus fumigatus, and Epstein-Barr virus (EBV) and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA., Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood. Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis, Salmonella enterica, Aspergillus fumigatus, and Epstein-Barr virus (EBV) and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA. The PCR cycle threshold (CT) was used to measure amplifiable cfDNA. In spiked samples, the median CT values for M. tuberculosis, S. enterica, and EBV cfDNA were significantly lower in blood collected in K2EDTA tubes than those in Streck and PAXgene blood collection tubes, and they were was significantly lower in urine preserved with EDTA (EDTA-urine) than in urine preserved with Streck reagent (Streck-urine). Blood and urine samples from TB patients preserved with K2EDTA and Tris-EDTA, respectively, showed significantly lower median M. tuberculosis CT values than with the Streck blood collection tube and Streck urine preservative. Processing delay increased the median pathogen CT values for Streck and PAXgene but not K2EDTA blood samples and for urine preserved with Streck reagent but not EDTA. Double-spin compared with single-spin plasma separation increased the median pathogen CT regardless of blood collection tube. No differences were observed between whole urine and supernatant and between fresh and thawed plasma and urine after 24 weeks at –80°C. Larger plasma and urine volumes in contrived and patient samples showed a significantly lower median M. tuberculosis CT. These findings suggest that large-volume single-spin K2EDTA-plasma and EDTA-whole urine with up to a 24-h processing delay may optimize pcfDNA detection.

Introduction Pneumonia is the leading cause of death globally in children. Supplemental oxygen reduces mortality but is not available in many low-resource settings. Inadequate power supply to drive oxygen concentrators is a major contributor to this failure. The objectives of our study were to (a) assess the availability of therapeutic oxygen; (b) evaluate the reliability of the electrical supply; and (c) investigate the effects of suboptimal oxygen delivery on patient outcomes in selected healthcare facilities in rural Kenya. Materials and methods A cross-sectional descriptive study on oxygen availability and descriptive case series of Kenyan children and youth hospitalized with hypoxemia. Results Two of 11 facilities had no oxygen equipment and nine facilities had at least one concentrator or cylinder. Facilities had a median of seven power interruptions per week (range: 2-147). The median duration of the power outage was 17 minutes and the longest was more than 6 days. The median proportion of time without power was out 7% (range: 1%-58%). Fifty-seven patients hospitalized with hypoxemia (median oxygen saturation 85% [interquartile range {IQR}: 82-87]) were included in our case series. Patients received supplemental oxygen for a median duration of 4.6 hours (IQR: 3.0-7.8). Eighteen patients (32%) faced an oxygen interruption of the median duration of 11 minutes (IQR: 9-20). A back-up cylinder was used in 5/18 (28%) cases. The case fatality rate was 11/57 (19%). Conclusion Mortality due to hypoxemia remains unacceptably high in low-resource healthcare facilities and may be associated with oxygen insecurity, related to lack of equipment and/or reliable power.

To help address the limitations of operating conventional microbiological culture incubators in low resource environments, a new incubator design was developed and tested to meet the requirements of operation in laboratories without reliable power (power outages up to 12 contiguous hours) or climate control (ambient indoor temperatures from 5 °C to 45 °C). The device is designed to enable adherence to incubation temperatures recommended for growth detection, identification, and drug susceptibility testing (DST) of human pathogenic bacteria. During power outages, stable temperatures are maintained in the device's internal sample compartment by employing phase change material (PCM) as a bi-directional thermal battery to maintain incubation temperature. Five prototypes were tested in a laboratory setting using environmental test chambers and programmable power supplies, and three were field tested in the Lao PDR in situations of intended use. The prototypes successfully held their temperature to within ±1 °C in both laboratory environmental chamber testing as well as during the field test. The results indicate that the device will maintain stable culture temperatures across periods of intermittent power supply, while enabling normal workflow of this could greatly increase the availability of microbiological culture for diagnosis and antimicrobial resistance (AMR) monitoring.

Author(s): Wood, Aaron; Barizuddin, Syed; Darr, Charles M; Mathai, Cherian J; Ball, Alexey; Minch, Kyle; Somoskovi, Akos; Hamasur, Beston; Connelly, John T; Weigl, Bernhard; Andama, Alfred; Cattamanchi, Adithya; Gangopadhyay, Keshab; Bok, Sangho; Gangopadhyay, Shubhra | Abstract: BACKGROUND:Timely diagnosis of tuberculosis disease is critical for positive patient outcomes, yet potentially millions go undiagnosed or unreported each year. Sputum is widely used as the testing input, but limited by its complexity, heterogeneity, and sourcing problems. Finding methods to interrogate noninvasive, non-sputum clinical specimens is indispensable to improving access to tuberculosis diagnosis and care. In this work, economical plasmonic gratings were used to analyze tuberculosis biomarker lipoarabinomannan (LAM) from clinical urine samples by single molecule fluorescence assay (FLISA) and compared with gold standard sputum GeneXpert MTB/ RIF, culture, and reference ELISA testing results. METHODS AND FINDINGS:In this study, twenty sputum and urine sample sets were selected retrospectively from a repository of HIV-negative patient samples collected before initiation of anti-tuberculosis therapy. GeneXpert MTB/RIF and culture testing of patient sputum confirmed the presence or absence of pulmonary tuberculosis while all patient urines were reference ELISA LAM-negative. Plasmonic gratings produced by low-cost soft lithography were bound with anti-LAM capture antibody, incubated with patient urine samples, and biotinylated detection antibody. Fluorescently labeled streptavidin revealed single molecule emission by epifluorescence microscope. Using a 1 fg/mL baseline for limit of detection, single molecule FLISA demonstrated good qualitative agreement with gold standard tests on 19 of 20 patients, including accurately predicting the gold-standard-negative patients, while one gold-standard-positive patient produced no observable LAM in urine. CONCLUSIONS:Single molecule FLISA by plasmonic grating demonstrated the ability to quantify tuberculosis LAM from complex urine samples of patients from a high endemic setting with negligible interference from the complex media itself. Moreover, agreement with patient diagnoses by gold standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively.

Access to therapeutic oxygen remains a challenge in the effort to reduce pneumonia mortality among children in low- and middle-income countries. The use of oxygen concentrators is common, but their effectiveness in delivering uninterrupted oxygen is gated by reliability of the power grid. Often cylinders are employed to provide continuous coverage, but these can present other logistical challenges. In this study, we examined the use of a novel, low-pressure oxygen storage system to capture excess oxygen from a concentrator to be delivered to patients during an outage. A prototype was built and tested in a non-clinical trial in Jinja, Uganda. The trial was carried out at Jinja Regional Referral Hospital over a 75-day period. The flow rate of the unit was adjusted once per week between 0.5 and 5 liters per minute. Over the trial period, 1284 power failure episodes with a mean duration of 3.1 minutes (range 0.08 to 1720 minutes) were recorded. The low-pressure system was able to deliver oxygen over 56% of the 4,295 power outage minutes and cover over 99% of power outage events over the course of the study. These results demonstrate the technical feasibility of a method to extend oxygen availability and provide a basis for clinical trials.

Tuberculosis (TB) recently surpassed HIV/AIDS as the deadliest infectious disease. Despite some advancements in technologies and programs, TB remains a global health epidemic that, without significant gains in better diagnostic and management, will not abate. This review describes the molecular diagnostic gaps, needs, and potential solutions for improving access to diagnosis and management of patients with drug susceptible and drug resistant TB. The ultimate goal is to improve laboratory diagnostics and better individualized managementof detected TB, and make an important contribution toward eliminating this global epidemic. RATIONALE: Tuberculosis persists as one of the world's most deadly infectious disease. To make substantive progress toward TB elimination and end the global tuberculosis epidemic by 2035, innovative approaches and technologies are essential. Among the various related and complementary scientific and clinical milestones toward elimination, including vaccine development and new therapeutic regimens, one of the greatest challenges is to address the gaps in existing diagnostics. Current diagnostic technologies fall short in meeting the complex nature of the disease and its epidemiologic profile. This review concentrates and outlines the priorities for development of laboratory diagnostic tools for screening and diagnosing TB. APPROACH: The approaches and priorities described in this manuscript are based on identified gaps of current laboratory diagnosis of different clinical forms of TB. Closing these gaps will enable national programs to more efficiently diagnose TB, monitor treatment efficacy, and control drug resistance. Prioritization is based on technologies that address facilitate a more patient-centered approach, and are appropriate for low-resource and high-burden TB settings. New diagnostics must complement current or anticipated developments elsewhere and accelerate increased coverage, quality, and impact at the lower levels of the healthcare system.

SUMMARY Mycobacteria are the causative organisms for diseases such as tuberculosis (TB), leprosy, Buruli ulcer, and pulmonary nontuberculous mycobacterial disease, to name the most important ones. In 2015, globally, almost 10 million people developed TB, and almost half a million patients suffered from its multidrug-resistant form. In 2016, a total of 9,287 new TB cases were reported in the United States. In 2015, there were 174,608 new case of leprosy worldwide. India, Brazil, and Indonesia reported the most leprosy cases. In 2015, the World Health Organization reported 2,037 new cases of Buruli ulcer, with most cases being reported in Africa. Pulmonary nontuberculous mycobacterial disease is an emerging public health challenge. The U.S. National Institutes of Health reported an increase from 20 to 47 cases/100,000 persons (or 8.2% per year) of pulmonary nontuberculous mycobacterial disease among adults aged 65 years or older throughout the United States, with 181,037 national annual cases estimated in 2014. This review describes contemporary methods for the laboratory diagnosis of mycobacterial diseases. Furthermore, the review considers the ever-changing health care delivery system and stresses the laboratory's need to adjust and embrace molecular technologies to provide shorter turnaround times and a higher quality of care for the patients who we serve.

To realize the most benefit from multidrug-resistant tuberculosis (MDR-TB) screening, all nucleic acid amplification test (NAAT)-positive respiratory specimens should be universally tested. Once an MDR-TB diagnosis is established, additional testing is warranted to provide details about the detected mutations. The lab-on-chip technology described by A. M. Cabibbe et al. (J Clin Microbiol 53:3876–3880, 2015, http://dx.doi.org/10.1128/JCM.01824-15 ) potentially provides this much needed information.

The performance of urine Xpert MTB/RIF Ultra (Xpert Ultra) for pulmonary TB diagnosis is unknown. HIV-positive and HIV-negative adults were enrolled at two health facilities in Kampala, Uganda. We compared the accuracy of urine Xpert Ultra and Determine TB-LAM in reference to sputum-based testing (positive Xpert MTB/RIF or culture), and assessed incremental yield. Urine Xpert Ultra had low sensitivity (17.2%, 95% CI 12.3-23.2) but high specificity (98.1%, 95% CI 94.4-99.6). Sensitivity reached 50.0% (95% CI 28.2-71.8) among HIV-positive patients with CD4

An urgent question that needs to be addressed rapidly by the mycobacteriology laboratory is whether Mycobacterium tuberculosis complex or NTM is involved. NAA assays are excellent tools for the purpose, and can be used directly on the clinical specimens of patients suspected of having mycobacterial disease, allowing same-day reporting of results. The CDC recommends using both liquid and solid media for growth detection of mycobacteria to decrease the time to detection and to increase the yield of growth detection. DNA sequencing of variable genomic regions offers a rapid, accurate, and relatively inexpensive method for the identification of mycobacteria.

Summary Lack of laboratory capacity in high-burden countries presents a significant barrier in providing diagnosis and treatment to patients infected with HIV/AIDS, tuberculosis (TB), and malaria. Strengthening laboratory services in these settings via implementation of novel diagnostic tests is especially indispensable to battle against TB and MDR/XDR-TB. Novel TB diagnostic assays have to be applicable to field conditions, affordable and accessible to all patients at different levels of the tiered laboratory network. A functional laboratory network should be based on well-developed diagnostic platforms that can provide not only timely and adequate diagnosis but are simple to use, and easy to implement and sustain. The article is overviewing the latest developments of TB laboratory diagnostics such as smear microscopy, rapid growth detection and susceptibility testing and rapid molecular testing with focus on performance and applicability to diagnostic needs at different levels of the diagnostic network.

Differentiation of Mycobacterium tuberculosis complex (MTC) species is important for patient management. We developed a genomic deletion assay based on multiplex polymerase chain reaction with melting temperature analysis that correctly identified 124 (96%) of 129 MTC isolates. This assay is a fast single-tube method to differentiate members of MTC.

The automated GenoType MTBC assay was evaluated for the ability to detect and identify members of the Mycobacterium tuberculosis complex. In addition to 35 reference strains and 157 clinical isolates, performance of this assay was tested directly on 79 smear-positive clinical specimens. The assay proved as accurate as the reference deletion analysis for all 192 isolates and detected and identified M. tuberculosis complex members in 93.2% of the specimens containing the M. tuberculosis complex.

IS The laboratory diagnosis of tuberculosis must be accelerated and expanded not only in response to the changes in patient populations, but also in fulfillment of the need for shorter turnaround times and increased accuracy that enables conservation of limited healthcare resources. This review describes novel and nonconventional methods for the diagnosis of tuberculosis. 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins

We describe the case of a 78-year-old male with bladder cancer who developed a mediastinal mass after intravesical immunotherapy with live Mycobacterium bovis BCG. The clinical diagnosis was mediastinal tumor suggestive for lymphoma. However, cultures of the biopsy specimens grew an acid-fast organism, which was identified as M. bovis BCG. To the best of our knowledge, this is the first reported case in which a post-instillation BCG infection induced a mediastinal mass that mimicked a tumor in a patient with bladder cancer.

Background: Mycobacterium tuberculosis strains belonging to the W-Beijing family have received broad clinical and public health attention because of their rapid worldwide spread and their frequent association with outbreaks, multidrug resistance, and treatment failures and relapses. Methods: The present study examined a large number of multidrug-resistant strain-W isolates (isolates of 29 patients) by susceptibility testing for pyrazinamide (PZA) using the reference BACTEC 460TB method (Becton Dickinson Diagnostic Instrument Systems; Sparks, MD) and also by DNA sequencing of the pncA gene. Results: We found that despite of the presence of a strain W-specific Thr47Ala in the pncA gene, all strains showed susceptibility to PZA in the reference BACTEC 460TB system due to their higher minimum inhibitory concentrations (relative to BACTEC 460TB PZA-susceptible strains). Conclusions: Our results suggest that the current radiometric reference method cannot reproducibly detect PZA resistance in patients infected with W-Beijing strains. Therefore, PZA susceptibility testing should instead be based on analysis of the pncA gene for resistance-associated mutations.

Testing for susceptibility to pyrazinamide (PZA) and analysis of the pncA gene sequences of 423 Mycobacterium tuberculosis complex isolates have revealed a unique silent nucleotide substitution that enables the rapid identification of “M. canettii” (proposed name). Moreover, the lack of a defined mutation within the pncA gene strongly suggests that an alternative mechanism is responsible for PZA resistance. Our results indicate that DNA sequencing of the pncA gene has the potential to shorten the turnaround time and increase the accuracy of PZA susceptibility testing of the M. tuberculosis complex. The Mycobacterium tuberculosis complex (MTB complex) consists of the closely related organisms M. tuberculosis, M. africanum, M. bovis, M. bovis BCG, M. caprae, M. microti, M. pinnipedii, the dassie bacillus, and “M. canettii” (proposed name) (3, 5, 6, 26). Although the members of the complex may differ in their epidemiologies, host spectra, geographic ranges, pathogenicities, and antituberculosis drug susceptibilities and although they display different phenotypic characteristics by conventional biochemical tests, they show high degrees of genetic homogeneity (10, 25). Conventional phenotypic methods for the identification of the members of the MTB complex are laborious and timeconsuming, and they require a large biomass (13). Thus, molecular methods (such as DNA sequencing of the oxyR, pncA, gyrB ,o rhsp65 gene; analysis of spacers between direct repeats in the direct repeat region; and deletion analysis of the regions of difference [RD]) provide a more rapid and accurate approach to the differentiation of the members of the MTB complex (8, 9, 16, 23, 24).

To realize the most benefit from multidrug-resistant tuberculosis (MDR-TB) screening, all nucleic acid amplification test (NAAT)-positive respiratory specimens should be universally tested. Once an MDR-TB diagnosis is established, additional testing is warranted to provide details about the detected mutations. The lab-on-chip technology described by A. M. Cabibbe et al. (J Clin Microbiol 53:3876-3880, 2015, http://dx.doi.org/10.1128/JCM.01824-15) potentially provides this much needed information.

Bronchial asthma is one of the most common maternal diseases complicating pregnancy. We assessed the risks of congenital abnormalities in a case-control population-based analysis using the dataset of the Hungarian Case-Control Surveillance of Congenital Abnormalities between 1980 and 1996. Of the 22,843 cases with congenital abnormalities, 511 (2.2%) had mothers with bronchial asthma, while of the 38,151 matched control subjects without congenital abnormalities 757 (2.0%) had mothers with bronchial asthma (unadjusted prevalence odds ratios [POR] 1.2; 95% CI: 1.0-1.3). In all mothers with bronchial asthma, a higher incidence of respiratory tract infections and higher drug intake could be observed. In the case group of medically recorded bronchial asthma, a slightly increased risk for club foot has been revealed. However, this weak association could be explained by the higher proportion of preterm births in this group. The main limitation of the analysis was that at the time of data collection only a small proportion of pregnant mothers were using anti-asthma medications recommended by the actual guidelines.

In Hungary the incidence of tuberculosis among the homeless population was 676 per 100,000 in 2002. Sixty-nine percent (140 patients) of all homeless tuberculosis patients were notified in Budapest (the capital). Therefore, a retrospective study that included 66 homeless tuberculosis patients notified in Budapest in 2002 was conducted to determine the rate of recent transmission of the disease and medical risk factors and to identify transmission pathways by means of conventional and molecular epidemiologic methods. IS 6110 DNA fingerprinting revealed that 71.2% of the isolates could be clustered. Thirty-four (51.5%) patients belonged to five major clusters (size, from 4 to 11 individuals), and 13 (19.7%) belonged to six smaller clusters. Additional analysis of patient records found that 2 (18%) of the 11 patients in cluster A, 3 (37.5%) of the 8 patients in cluster B, and 2 (33%) of the 6 patients in cluster C were residents of the same three homeless shelters during the diagnosis of tuberculosis. Review of the database of the National Tuberculosis Surveillance Center (NTSC) revealed that 21.2% of the cases have not been reported to the NTSC. These findings indicate that the screening and treatment of tuberculosis among the homeless need to be strengthened and also warrant the review of environmental control steps in public shelters. Improvement of adherence of clinicians to surveillance reporting regulations is also necessary.

Background: Pyrazinamide is a paradoxical frontline tuberculosis drug characterized by high in vivo steril- izing activity but poor in vitro activity. This separation in pyrazinamide activity reflects differences between the in vivo tissue environment and in vitro culture conditions. The well-known acid pH requirement for pyrazin- amide activity was discovered previously based on such reasoning but does not completely explain the discrepancy between in vivo and in vitro activity of pyrazinamide. This study examined the effect of iron, which could potentially be elevated in local inflammatory lesions, on pyrazinamide activity in vitro. Materials and methods: The effect of iron on the activity of pyrazinamide or its active derivative pyrazinoic acid against Mycobacterium tuberculosis was assessed in liquid medium in a drug exposure assay or in solid medium with pyrazinamide plus iron or pyrazinamide alone. The effect of iron on pyrazinamide or pyrazinoic acid was expressed as percentage of growth inhibition. Results: We have shown that iron enhances the activity of pyrazinamide and pyrazinoic acid against M. tuberculosis in both liquid and solid media at acid pH 5.6. Iron enhanced the activity of pyrazinoic acid but not pyrazinamide against the naturally pyrazinamide-resistant Mycobacterium bovis BCG. Other metal ions such as magnesium, calcium and zinc did not enhance the activity of pyrazinamide or pyrazinoic acid. Conclusions: Iron increased the activity of pyrazinamide or pyrazinoic acid against M. tuberculosis in vitro. These findings may have implications for the study of mechanism of action of pyrazinamide and possible iron supplement for improving the activity of pyrazinamide.

Acquired clofazimine and bedaquiline cross-resistance in a patient due to mutation in the M. tuberculosis Rv0678 gene http://ow.ly/Cnkk7