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The World Health Organization's (WHO) "End TB" strategy calls for development and implementation of novel tuberculosis (TB) diagnostics. Sputum-based diagnostics are challenging to implement and often less sensitive in high-priority populations. Nonsputum, biomarker-based tests may facilitate TB testing at lower levels of the healthcare system, accelerate treatment initiation, and improve outcomes. We provide guidance on the design of diagnostic accuracy studies evaluating nonsputum, biomarker-based tests within the context of WHO's target product profile for such tests. Study designs should account for the intended use when choosing the study population, setting, and reference standards. Although adults with respiratory symptoms may be an initial target population, other high-priority populations regardless of symptoms-including people living with human immunodeficiency virus, those unable to produce sputum samples or with extrapulmonary TB, household contacts, and children-should be considered. Studies beyond diagnostic accuracy that evaluate feasibility and population-level impacts are also needed. A biomarker-based diagnostic may be critical to ending the TB epidemic, but requires appropriate validation before implementation., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Tests that can replace sputum smear microscopy have been identified as a top priority diagnostic need for tuberculosis by the World Health Organization. High-quality evidence on diagnostic accuracy for tests that may meet this need is an essential requirement to inform decisions about policy and scale-up. However, test accuracy studies are often of low and inconsistent quality and poorly reported, leading to uncertainty about true test performance. Here we provide guidance for the design of diagnostic test accuracy studies of sputum smear-replacement tests. Such studies should have a cross-sectional or cohort design, enrolling either a consecutive series or a random sample of patients who require evaluation for tuberculosis. Adults with respiratory symptoms are the target population. The reference standard should at a minimum be a single, automated, liquid culture, but additional cultures, follow-up, clinical case definition, and specific measures to understand discordant results should also be included. Inclusion of smear microscopy and Xpert MTB/RIF (or MTB/RIF Ultra) as comparators is critical to allow broader comparability and generalizability of results, because disease spectrum can vary between studies and affects relative test performance. Given the complex nature of sputum (the primary specimen type used for pulmonary TB), careful design and reporting of the specimen flow is essential. Test characteristics other than accuracy (such as feasibility, implementation considerations, and data on impact on patient, population and health systems outcomes) are also important aspects., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Novel accurate tests are needed that identify individuals infected with Mycobacterium tuberculosis who have incipient disease and are likely to develop clinical tuberculosis (TB) in the near future to allow for targeted preventive treatment beyond the current risk groups. Recently, a target product profile was developed that outlines the minimal and optimal characteristics for such an incipient TB test. We describe an evaluation framework for generating evidence to inform the development of policy guidance for the use of such a new test by the World Health Organization. Two research objectives are addressed. 1) The predictive ability of an incipient TB test should be assessed in clinical evaluation studies that include the intended target population and follow-up of sufficient duration to observe whether individuals do or do not progress to clinical TB disease. 2) Studies are needed to evaluate the test under routine programmatic conditions and measure its impact on patient- or health-system-important outcomes. For both research objectives, study designs, methods and analysis are described, with the intent to inform the clinical development plans of test manufacturers, researchers and funders., Competing Interests: Conflict of interest: C.M. Denkinger: FIND (Foundation for Innovative New Diagnostics) is a not-for-profit foundation, whose mission is to find diagnostic solutions to overcome diseases of poverty in low- and middle-income countries (LMICs). It works closely with the private and public sectors and receives funding from some of its industry partners. It has organisational firewalls to protect it against any undue influences in its work or the publication of its findings. All industry partnerships are subject to review by an independent Scientific Advisory Committee or another independent review body, based on due diligence, target product profiles and public sector requirements. FIND catalyses product development, leads evaluations, takes positions and accelerates access to tools identified as serving its mission. It provides indirect support to industry (e.g. access to open specimen banks, a clinical trial platform, technical support, expertise, laboratory capacity strengthening in LMICs) to facilitate the development and use of products in these areas. FIND also supports the evaluation of prioritised assays and the early stages of implementation of WHO-approved (guidance and prequalification) assays using donor grants. In order to carry out test validations and evaluations, FIND has product evaluation agreements with several private sector companies, which strictly define its independence and neutrality vis-a-vis the companies whose products get evaluated, and describe roles and responsibilities., (The content of this work is copyright of the authors or their employers. Design and branding are copyright ©ERS 2018.)

Drug-resistant tuberculosis poses a persistent public health threat. The ReSeqTB platform is a collaborative, curated knowledgebase, designed to standardize and aggregate global Mycobacterium tuberculosis complex (MTBC) variant data from whole genome sequencing (WGS) with phenotypic drug susceptibility testing (DST) and clinical data. We developed a unified analysis variant pipeline (UVP) ( https://github.com/CPTR-ReSeqTB/UVP ) to identify variants and assign lineage from MTBC sequence data. Stringent thresholds and quality control measures were incorporated in this open source tool. The pipeline was validated using a well-characterized dataset of 90 diverse MTBC isolates with conventional DST and DNA Sanger sequencing data. The UVP exhibited 98.9% agreement with the variants identified using Sanger sequencing and was 100% concordant with conventional methods of assigning lineage. We analyzed 4636 publicly available MTBC isolates in the ReSeqTB platform representing all seven major MTBC lineages. The variants detected have an above 94% accuracy of predicting drug based on the accompanying DST results in the platform. The aggregation of variants over time in the platform will establish confidence-graded mutations statistically associated with phenotypic drug resistance. These tools serve as critical reference standards for future molecular diagnostic assay developers, researchers, public health agencies and clinicians working towards the control of drug-resistant tuberculosis.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is caused by autosomal dominant germline mutations in the fumarate hydratase (FH) gene and is characterized by cutaneous leiomyomas, uterine leiomyomas and aggressive renal malignancies. We conducted a retrospective chart review to characterize the patients referred to our Regional Genetics Program for assessment of HLRCC from 2004 to mid-2016. Forty-eight of 69 (69.5%) referred individuals were positive for a pathogenic or likely pathogenic variant in FH; they had an average age of 39.1 years. There were 11 different FH variants among them. As expected, the most sensitive indications for a positive genetic test were papillary renal cell carcinoma (RCC) at a young age (5/5; 100%) and multiple cutaneous leiomyomas (18/19; 95%). However, only twenty-two of 48 (46%) individuals with a positive molecular test had cutaneous leiomyomas, which is considerably lower than previously reported and supports the likelihood of ascertainment bias in previous reports. Notably, we have experience with 1 large family in which there were no cutaneous leiomyomas across a large age range. We confirm that multiple cutaneous leiomyomas and papillary RCCs at a young age have a high positive predictive value for a molecular diagnosis of HLRCC, but that cutaneous leiomyomas are less prevalent in HLRCC than previously understood, and therefore the condition is likely to be under-ascertained. Our understanding of the phenotypic spectrum of HLRCC is still evolving.

Background: In many countries, regular monitoring of the emergence of resistance to anti-tuberculosis drugs is hampered by the limitations of phenotypic testing for drug susceptibility. We therefore evaluated the use of genetic sequencing for surveillance of drug resistance in tuberculosis., Methods: Population-level surveys were done in hospitals and clinics in seven countries (Azerbaijan, Bangladesh, Belarus, Pakistan, Philippines, South Africa, and Ukraine) to evaluate the use of genetic sequencing to estimate the resistance of Mycobacterium tuberculosis isolates to rifampicin, isoniazid, ofloxacin, moxifloxacin, pyrazinamide, kanamycin, amikacin, and capreomycin. For each drug, we assessed the accuracy of genetic sequencing by a comparison of the adjusted prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing., Findings: Isolates were taken from 7094 patients with tuberculosis who were enrolled in the study between November, 2009, and May, 2014. In all tuberculosis cases, the overall pooled sensitivity values for predicting resistance by genetic sequencing were 91% (95% CI 87-94) for rpoB (rifampicin resistance), 86% (74-93) for katG, inhA, and fabG promoter combined (isoniazid resistance), 54% (39-68) for pncA (pyrazinamide resistance), 85% (77-91) for gyrA and gyrB combined (ofloxacin resistance), and 88% (81-92) for gyrA and gyrB combined (moxifloxacin resistance). For nearly all drugs and in most settings, there was a large overlap in the estimated prevalence of drug resistance by genetic sequencing and the estimated prevalence by phenotypic testing., Interpretation: Genetic sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in tuberculosis in resource-poor countries. Before its widespread adoption for surveillance purposes, there is a need to standardise DNA extraction methods, recording and reporting nomenclature, and data interpretation., Funding: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development., (© 2018 World Health Organization; licensee Elsevier. This is an Open Access article published under the CC BY 3.0 IGO license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In any use of this article, there should be no suggestion that WHO endorses any specific organisation, products or services. The use of the WHO logo is not permitted. This notice should be preserved along with the article's original URL.)

Nanopores have become a subject of interest in the scientific community due to their potential uses in nanometer-scale laboratory and research applications, including infectious disease diagnostics and DNA sequencing. Additionally, they display behavioral similarity to molecular and cellular scale physiological processes. Recent advances in information theory have made it possible to probe the information dynamics of nonlinear stochastic dynamical systems, such as autonomously fluctuating nanopore systems, which has enhanced our understanding of the physical systems they model. We present the results of local (LER) and specific entropy rate (SER) computations from a simulation study of an autonomously fluctuating nanopore system. We learn that both metrics show increases that correspond to fluctuations in the nanopore current, indicating fundamental changes in information generation surrounding these fluctuations., Competing Interests: The authors declare no conflict of interest.

A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence.Raw genotype-phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance.We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6-90.9%), while for isoniazid it was 78.2% (77.4-79.0%) and their specificities were 96.3% (95.7-96.8%) and 94.4% (93.1-95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1-70.6%) for capreomycin to 88.2% (85.1-90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1-92.5%) for moxifloxacin to 99.5% (99.0-99.8%) for amikacin.This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

Au-Fe x O y composite nanoparticles (NPs) are of great technological interest due to their combined optical and magnetic properties. However, typical syntheses are neither simple nor ecologically friendly, creating a challenging situation for process scale-up. Here we describe conditions for preparing Au-Fe x O y NPs in aqueous solutions and at ambient temperatures, without resorting to solvents or amphiphilic surfactants with poor sustainability profiles. These magnetic gold nanoclusters (MGNCs) are prepared in practical yields with average sizes slightly below 100 nm, and surface plasmon resonances that extend to near-infrared wavelengths, and sufficient magnetic moment (up to 6 emu g -1 ) to permit collection within minutes by handheld magnets. The MGNCs also produce significant photoluminescence when excited at 488 nm. Energy dispersive X-ray (EDX) analysis indicates a relatively even distribution of Fe within the MGNCs, as opposed to a central magnetic core.

Patients with progeroid syndromes such as mandibuloacral dysplasia, type B (MADB) and restrictive dermopathy (RD) harbor mutations in zinc metalloproteinase (ZMPSTE24), an enzyme essential for posttranslational proteolysis of prelamin A to form mature lamin A. Dermal fibroblasts from these patients show increased nuclear dysmorphology and reduced proliferation; however, the efficacy of various pharmacological agents in reversing these cellular phenotypes remains unknown. In this study, fibroblasts from MADB patients exhibited marked nuclear abnormalities and reduced proliferation that improved upon treatment with rapamycin and dimethylsulfoxide but not with other agents, including farnesyl transferase inhibitors. Surprisingly, fibroblasts from an RD patient with a homozygous null mutation in ZMPSTE24 , resulting in exclusive accumulation of prelamin A with no lamin A on immunoblotting of cellular lysate, exhibited few nuclear abnormalities and near-normal cellular proliferation. An unbiased proteomic analysis of the cellular lysate from RD fibroblasts revealed a lack of processing of vimentin, a cytoskeletal protein. Interestingly, the assembly of the vimentin microfibrils in MADB fibroblasts improved with rapamycin and dimethylsulfoxide. We conclude that rapamycin and dimethylsulfoxide are beneficial for improving nuclear morphology and cell proliferation of MADB fibroblasts. Data from a single RD patient's fibroblasts also suggest that prelamin A accumulation by itself might not be detrimental and requires additional alterations at the cellular level to manifest the phenotype.

Drug-resistant tuberculosis (DR-TB) poses a major threat to control of tuberculosis worldwide. Diagnosis and treatment of DR-TB are considerably more difficult than for drug-susceptible tuberculosis (TB) and require higher level infrastructure and proficiency from laboratory specialists and clinicians. The World Health Organization (WHO) End TB Strategy calls for early diagnosis and initiation of appropriate treatment of all persons of all ages with any form of drug-susceptible TB or DR-TB. This requires ensuring access to WHO-recommended rapid diagnostics and universal drug susceptibility testing (DST) for all persons with signs and symptoms of TB. There are a number of laboratory tools available for diagnosis of DR-TB, including phenotypic culture-based DST as well as molecular methods. Optimal and complementary use of the available diagnostic tools at the different levels of the tiered network of TB laboratories, as well as correct interpretation of the diagnostic results provided by them is critical for accurate and timely diagnosis of DR-TB thus enabling effective treatment and care of patients., Competing Interests: Conflict of interest statement: The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Background: Pyrazinamide and fluoroquinolones are essential antituberculosis drugs in new rifampicin-sparing regimens. However, little information about the extent of resistance to these drugs at the population level is available., Methods: In a molecular epidemiology analysis, we used population-based surveys from Azerbaijan, Bangladesh, Belarus, Pakistan, and South Africa to investigate resistance to pyrazinamide and fluoroquinolones among patients with tuberculosis. Resistance to pyrazinamide was assessed by gene sequencing with the detection of resistance-conferring mutations in the pncA gene, and susceptibility testing to fluoroquinolones was conducted using the MGIT system., Findings: Pyrazinamide resistance was assessed in 4972 patients. Levels of resistance varied substantially in the surveyed settings (3·0-42·1%). In all settings, pyrazinamide resistance was significantly associated with rifampicin resistance. Among 5015 patients who underwent susceptibility testing to fluoroquinolones, proportions of resistance ranged from 1·0-16·6% for ofloxacin, to 0·5-12·4% for levofloxacin, and 0·9-14·6% for moxifloxacin when tested at 0·5 μg/mL. High levels of ofloxacin resistance were detected in Pakistan. Resistance to moxifloxacin and gatifloxacin when tested at 2 μg/mL was low in all countries., Interpretation: Although pyrazinamide resistance was significantly associated with rifampicin resistance, this drug may still be effective in 19-63% of patients with rifampicin-resistant tuberculosis. Even though the high level of resistance to ofloxacin found in Pakistan is worrisome because it might be the expression of extensive and unregulated use of fluoroquinolones in some parts of Asia, the negligible levels of resistance to fourth-generation fluoroquinolones documented in all survey sites is an encouraging finding. Rational use of this class of antibiotics should therefore be ensured to preserve its effectiveness., Funding: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development., (Copyright © 2016 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY license. Published by Elsevier Ltd.. All rights reserved.)

Latent tuberculosis infection (LTBI) is characterised by the presence of immune responses to previously acquired Mycobacterium tuberculosis infection without clinical evidence of active tuberculosis (TB). Here we report evidence-based guidelines from the World Health Organization for a public health approach to the management of LTBI in high risk individuals in countries with high or middle upper income and TB incidence of <100 per 100 000 per year. The guidelines strongly recommend systematic testing and treatment of LTBI in people living with HIV, adult and child contacts of pulmonary TB cases, patients initiating anti-tumour necrosis factor treatment, patients receiving dialysis, patients preparing for organ or haematological transplantation, and patients with silicosis. In prisoners, healthcare workers, immigrants from high TB burden countries, homeless persons and illicit drug users, systematic testing and treatment of LTBI is conditionally recommended, according to TB epidemiology and resource availability. Either commercial interferon-gamma release assays or Mantoux tuberculin skin testing could be used to test for LTBI. Chest radiography should be performed before LTBI treatment to rule out active TB disease. Recommended treatment regimens for LTBI include: 6 or 9 month isoniazid; 12 week rifapentine plus isoniazid; 3-4 month isoniazid plus rifampicin; or 3-4 month rifampicin alone., (Copyright ©ERS 2015.)

Background: Novel diagnostics have been widely applied across human immunodeficiency virus (HIV) and tuberculosis prevention and treatment programs. To achieve the greatest impact, HIV and tuberculosis diagnostic programs must carefully plan and implement within the context of a specific healthcare system and the laboratory capacity., Methods: A workshop was convened in Cape Town in September 2014. Participants included experts from laboratory and clinical practices, officials from ministries of health, and representatives from industry., Results: The article summarizes best practices, challenges, and lessons learned from implementation experiences across sub-Saharan Africa for (1) building laboratory programs within the context of a healthcare system; (2) utilizing experience of clinicians and healthcare partners in planning and implementing the right diagnostic; and (3) evaluating the effects of new diagnostics on the healthcare system and on patient health outcomes., Conclusions: The successful implementation of HIV and tuberculosis diagnostics in resource-limited settings relies on careful consideration of each specific context., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

To accelerate the fight against tuberculosis, major diagnostic challenges need to be addressed urgently. Post-2015 targets are unlikely to be met without the use of novel diagnostics that are more accurate and can be used closer to where patients first seek care in affordable diagnostic algorithms. This article describes the efforts by the stakeholder community that led to the identification of the high-priority diagnostic needs in tuberculosis. Subsequently target product profiles for the high-priority diagnostic needs were developed and reviewed in a World Health Organization (WHO)-led consensus meeting. The high-priority diagnostic needs included (1) a sputum-based replacement test for smear-microscopy; (2) a non-sputum-based biomarker test for all forms of tuberculosis, ideally suitable for use at levels below microscopy centers; (3) a simple, low cost triage test for use by first-contact care providers as a rule-out test, ideally suitable for use by community health workers; and (4) a rapid drug susceptibility test for use at the microscopy center level. The developed target product profiles, along with complimentary work presented in this supplement, will help to facilitate the interaction between the tuberculosis community and the diagnostics industry with the goal to lead the way toward the post-2015 global tuberculosis targets., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

External quality assessment (EQA) for the Xpert MTB/RIF assay is part of the quality system required for clinical and laboratory practice. Five newly developed EQA panels that use different matrices, including a lyophilized sample (Vircell, Granada, Spain), a dried tube specimen (CDC), liquid (Maine Molecular Quality Control, Inc. [MMQCI], Scarborough, ME), artificial sputum (Global Laboratory Initiative [GLI]), and a dried culture spot (National Health Laboratory Services [NHLS]), were evaluated at 11 GeneXpert testing sites in South Africa. The panels comprised Mycobacterium tuberculosis complex (MTBC)-negative, MTBC-positive (including rifampin [RIF] susceptible and RIF resistant), and nontuberculosis mycobacterial material that was inactivated and safe for transportation. Twelve qualitative and quantitative variables were scored as acceptable (1) or unacceptable (0); the overall panel performance score for the Vircell, CDC, GLI, and NHLS panels was 9 of 12, while the MMQCI panel scored 6 of 12 (owing to the need for cold chain maintenance). All panels showed good compatibility with Xpert MTB/RIF testing, and none showed PCR inhibition. The use of a liquid or dry matrix did not appear to be a distinguishing criterion, as both matrices had reduced scores on insufficient volumes, a need for extra consumables, and the ability to transfer to the Xpert MTB/RIF cartridge. EQA is an important component of the quality system required for diagnostic testing programs, but it must be complemented by routine monitoring of performance indicators and instrument verification. This study aims to introduce EQA concepts for Xpert MTB/RIF testing and evaluates five potential EQA panels., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy in women under 40 years of age. We sequenced the exomes of six individuals from three families with SCCOHT. After discovering segregating deleterious germline mutations in SMARCA4 in all three families, we tested DNA from a fourth affected family, which also carried a segregating SMARCA4 germline mutation. All the familial tumors sequenced harbored either a somatic mutation or loss of the wild-type allele. Immunohistochemical analysis of these cases and additional familial and non-familial cases showed loss of SMARCA4 (BRG1) protein in 38 of 40 tumors overall. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. Additionally, the SCCOHT cell line BIN-67 had biallelic deleterious mutations in SMARCA4. Our findings identify alterations in SMARCA4 as the major cause of SCCOHT, which could lead to improvements in genetic counseling and new treatment approaches.

If tuberculosis (TB) is to be eliminated as a global health problem in the foreseeable future, improved detection of patients, earlier diagnosis and timely identification of rifampicin resistance will be critical. New diagnostics released in recent years have improved this perspective but they require investments in laboratory infrastructure, biosafety and staff specialisation beyond the means of many resource-constrained settings where most patients live. Xpert MTB/RIF, a new assay employing automated nucleic acid amplification to detect Mycobacterium tuberculosis, as well as mutations that confer rifampicin resistance, holds the promise to largely overcome these operational challenges. In this article we position Xpert MTB/RIF in today's TB diagnostic landscape and describe its additional potential as an adjunct to surveillance and surveys, taking into account considerations of pricing and ethics. In what could serve as a model for the future formulation of new policy on diagnostics, we trace the unique process by which the World Health Organization consulted international expertise and systematically assessed published evidence and freshly emerging experience from the field ahead of its endorsement of the Xpert MTB/RIF technology in 2010, summarise subsequent research findings and guidance on who to test and how, and provide perspectives on scaling up the new technology.

Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.

Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. Here, to identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide small interfering RNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to messenger RNA processing, interferon signalling, vesicle trafficking, cytoskeletal motor function and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, SMURF1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, SMURF1-deficient mice accumulate damaged mitochondria in the heart, brain and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines SMURF1 as a newly recognized mediator of both viral autophagy and mitophagy.

A knowledge, attitude, behaviour, and practice survey was conducted among labour migrants in Tajikistan to elucidate key factors influencing access to tuberculosis diagnosis and care both in their labour destination country and at home. 509 labour migrants were interviewed in Khaton and Rasht Valley regions in Tajikistan using a standardised questionnaire. In addition, in-depth interviews were conducted among ten tuberculosis patients who had recently worked abroad. The study showed that migrants have increased vulnerability to tuberculosis due to the working and living conditions in the destination country and that access to health services is limited due to their legal status or the high cost of health services abroad. The average knowledge of migrants regarding tuberculosis is low and misconceptions are frequent. In Tajikistan, although tuberculosis drugs are usually provided free of charge, tuberculosis diagnosis and ancillary treatment are charged, thus creating a significant financial burden for patients and their families. Improving the access of labour migrants to affordable early diagnosis and treatment in both host countries and Tajikistan is a priority.

A conserved insulin-like pathway modulates both aging and pathogen resistance in Caenorhabditis elegans. However, the specific innate effector functions that mediate this pathogen resistance are largely unknown. Autophagy, a lysosomal degradation pathway, plays a role in controlling intracellular bacterial pathogen infections in cultured cells, but less is known about its role at the organismal level. We examined the effects of autophagy gene inactivation on Salmonella enterica Serovar Typhimurium (Salmonella typhimurium) infection in 2 model organisms, Caenorhabditis elegans and Dictyostelium discoideum. In both organisms, genetic inactivation of the autophagy pathway increases bacterial intracellular replication, decreases animal lifespan, and results in apoptotic-independent death. In C. elegans, genetic knockdown of autophagy genes abrogates pathogen resistance conferred by a loss-of-function mutation, daf-2(e1370), in the insulin-like tyrosine kinase receptor or by over-expression of the DAF-16 FOXO transcription factor. Thus, autophagy genes play an essential role in host defense in vivo against an intracellular bacterial pathogen and mediate pathogen resistance in long-lived mutant nematodes.

Several protocols for isolation of mycobacteria from water exist, but there is no established standard method. This study compared methods of processing potable water samples for the isolation of Mycobacterium avium and Mycobacterium intracellulare using spiked sterilized water and tap water decontaminated using 0.005% cetylpyridinium chloride (CPC). Samples were concentrated by centrifugation or filtration and inoculated onto Middlebrook 7H10 and 7H11 plates and Lowenstein-Jensen slants and into mycobacterial growth indicator tubes with or without polymyxin, azlocillin, nalidixic acid, trimethoprim, and amphotericin B. The solid media were incubated at 32 degrees C, at 35 degrees C, and at 35 degrees C with CO(2) and read weekly. The results suggest that filtration of water for the isolation of mycobacteria is a more sensitive method for concentration than centrifugation. The addition of sodium thiosulfate may not be necessary and may reduce the yield. Middlebrook M7H10 and 7H11 were equally sensitive culture media. CPC decontamination, while effective for reducing growth of contaminants, also significantly reduces mycobacterial numbers. There was no difference at 3 weeks between the different incubation temperatures.

In 2006, the Australian Mycobacterium Reference Laboratory Network identified 905 bacteriologically confirmed cases of disease caused by members of the Mycobacterium tuberculosis complex. The annual reporting rate was 4.4 cases per 100,000 population. Of the 905 isolates, 903 were Mycobacterium tuberculosis and two were Mycobacterium bovis. Fourteen children aged under 10 years (male n = 5, female n = 9) had bacteriologically confirmed tuberculosis. A total of 100 (11.1%) isolates of M. tuberculosis were resistant to at least one first-line anti-tuberculosis agent. Resistance to at least H and R (defined as multi-drug resistant--MDR) was detected in 22 (2.4%) M. tuberculosis isolates. Of the 22 MDR-TB isolates, 17 were from the respiratory tract (sputum n = 11 bronchoscopy n = 5, nasogastric aspirate n = 1), three from lymph node, one from a sacral mass, and one sterile site fluid. Smear-positive specimens from the MDR-TB cases were found in sputum (n = 6), lymph node (n = 2), and one each of bronchoscopy and nasogastric aspirate specimens. The country of birth was known for all 100 cases with a drug-resistant isolate; 10 of whom were born in Australia. The 90 overseas-born cases with drug-resistant disease were from 27 countries. Two Australian-born cases had MDR-TB; one had worked extensively in the Philippines; the other was a contact of a known MDR-TB case. No cases of extensively drug-resistant TB (XDR-TB) were identified in 2006. However, an on-going review of laboratory data identified one case of XDR-TB in 2004.

This report is the consensus-based, agreed position of the participants in a workshop for experts in sputum smear microscopy organised in August 2005 by the International Union Against Tuberculosis and Lung Disease (The Union), and as such reflects their views, but not necessarily those of their respective organisations. The group reviewed current practices in sputum smear microscopy for tuberculosis (TB) and suggests considering certain changes in standard guidelines for the sputum collection strategy and the definition of sputum smear-positive TB. The Ziehl-Neelsen staining guidelines need to incorporate a wider error margin for widespread application under field conditions. Quality assurance is of utmost importance, and needs more commitment from National Tuberculosis Programmes and other health authorities. In particular, allocation of sufficient resources for rechecking and integration of laboratory supervision must be ensured. Countries must make better investments in the purchase of high quality microscopes and laboratory supplies. To address the human resource crisis, personnel without specific laboratory schooling can, in principle, be trained to respond to immediate needs for TB diagnostic microscopy services. Periodic reporting on acid-fast smear examinations is highly desirable for regular monitoring and a more balanced provision of supplies.

Context: Hereditary diffuse gastric cancer is caused by germline mutations in the epithelial cadherin (CDH1) gene and is characterized by an increased risk for diffuse gastric cancer and lobular breast cancer., Objective: To determine whether recurring germline CDH1 mutations occurred due to independent mutational events or common ancestry., Design, Setting, and Patients: Thirty-eight families diagnosed clinically with hereditary diffuse gastric cancer were accrued between November 2004 and January 2006 and were analyzed for CDH1 mutations as part of an ongoing study at the British Columbia Cancer Agency. Twenty-six families had at least 2 gastric cancer cases with 1 case of diffuse gastric cancer in a person younger than 50 years; 12 families had either a single case of diffuse gastric cancer diagnosed in a person younger than 35 years or multiple cases of diffuse gastric cancer diagnosed in persons older than 50 years., Main Outcome Measures: Classification of family members as carriers or noncarriers of CDH1 mutations. Haplotype analysis to assess recurring mutations for common ancestry was performed on 7 families from this study and 7 previously reported families with the same mutations., Results: Thirteen mutations (6 novel) were identified in 15 of the 38 families (40% detection rate). The 1137G>A splicing mutation and the 1901C>T (A634V) missense/splicing mutation occurred on common haplotypes in 2 families but on different haplotypes in a third family. The 2195G>A (R732Q) missense/splicing mutation occurred in 2 families on different haplotypes. The 2064-2065delTG mutation occurred on a common haplotype in 2 families. Two families from this study plus 2 additional families carrying the novel 2398delC mutation shared a common haplotype, suggesting a founder effect. All 4 families originate from the southeast coast of Newfoundland. Due to concentrations of lobular breast cancer cases, 2 branches of this family had been diagnosed as having hereditary breast cancer and were tested for BRCA mutations. Within these 4 families, the cumulative risk by age 75 years in mutation carriers for clinically detected gastric cancer was 40% (95% confidence interval [CI], 12%-91%) for males and 63% (95% CI, 19%-99%) for females and the risk for breast cancer in female mutation carriers was 52% (95% CI, 29%-94%)., Conclusions: Recurrent CDH1 mutations in families with hereditary diffuse gastric cancer are due to both independent mutational events and common ancestry. The presence of a founder mutation from Newfoundland is strongly supported.

Purpose: To correlate the clinical presentation and treatment outcome of Nocardia keratitis with the time to diagnosis, different species and with the drug sensitivity pattern., Method: Patients with Nocardia corneal ulcers were studied at a tertiary eye care center. Speciation of Nocardia isolates was done by sequencing the 16s rRNA gene. Clinical response to treatment was assessed by chart review., Results: Twenty one (65.3%) patients presented within 15 days of the onset of symptoms with typical clinical features of Nocardia keratitis, ie, a ring-like distribution of superficial infiltrates in a wreath pattern. Eight patients (25%) who presented after 15 days and within 30 days had an ulcer resembling fungal keratitis. N. Cyriacigeorgica (n = 11; 34.37%), N. asteroides (n = 9; 28%), N. farcinica (n = 7; 22%,) and N. Otitidiscaviarum (n = 5; 16%). All the species had 100% sensitivity to amikacin, sulphamethoxazole, imipenem and co-trimoxazole. Time to diagnosis of the infection was significantly associated with the different types of clinical presentation; those presenting early having the typical clinical picture (P = 0.004). Patients (73%) presenting within 15 days showed a highest recovery rate. (P = 0.045). The recovery time of the patients when compared with species showed those who were infected with N. cyriageorgica had a healing time of less than 15 days. Clinically, healing was faster when treated with 2% amikacin. Visual outcome improved in fourteen patients (44%) and sixteen (50%) patients remained the same (P = 0.0001)., Conclusion: Characteristic clinical picture of Nocardia is dependant on early presentation. It could be mistaken for fungal keratitis. Microbiological confirmation is important. Drug of choice for Nocardia keratitis is amikacin.

Autophagy is commonly observed in metazoan organisms during programmed cell death (PCD), but its function in dying cells has been unclear. We studied the role of autophagy in embryonic cavitation, the earliest PCD process in mammalian development. Embryoid bodies (EBs) derived from cells lacking the autophagy genes, atg5 or beclin 1, fail to cavitate. This defect is due to persistence of cell corpses, rather than impairment of PCD. Dying cells in autophagy gene null EBs fail to express the "eat-me" signal, phosphatidylserine exposure, and secrete lower levels of the "come-get-me" signal, lysophosphatidylcholine. These defects are associated with low levels of cellular ATP and are reversed by treatment with the metabolic substrate, methylpyruvate. Moreover, mice lacking atg5 display a defect in apoptotic corpse engulfment during embryonic development. We conclude that autophagy contributes to dead-cell clearance during PCD by a mechanism that likely involves the generation of energy-dependent engulfment signals.

The Australian Mycobacterium Reference Laboratory Network (AMRLN) collects and analyses laboratory data on new cases of disease caused by the Mycobacterium tuberculosis complex. In 2005, a total of 810 cases were identified by bacteriology; an annual reporting rate of 4.0 cases per 100,000 population. Isolates were identified as M. tuberculosis (n = 806), Mycobacterium africanum (n = 2) and Mycobacterium bovis (n = 2). Fifteen children aged under 10 years had bacteriologically-confirmed tuberculosis. Results of in vitro drug susceptibility testing were available for all 810 isolates for isoniazid (H), rifampicin (R), ethambutol (E), and pyrazinamide (Z). A total of 74 (9.1%) isolates of M. tuberculosis were resistant to at least one of these anti-tuberculosis agents. Resistance to at least H and R (defined as multi-drug resistance, MDR) was detected in 12 (1.5%) isolates; nine were from the respiratory tract (sputum n = 8, bronchoscopy n = 1). Of the 74 M. tuberculosis isolates resistant to at least one of the standard drugs, 67 (90.5%) were from new cases, 6 from previously treated cases, and no information was available on the remaining case. Eight were Australian-born, 65 were overseas-born, and the country of birth of one was unknown. Of the 65 overseas-born persons with drug resistant disease, 41 (63.1%) were from 4 countries; Vietnam (n = 16), Papua New Guinea (n = 10), the Philippines (n = 9), and India (n = 6). A retrospective review of AMRLN data on isolates collected between 2000 and 2005 found that none of 70 MDR-TB isolates met the new definition for extensively drug resistant TB (XDR-TB, i.e. MDR-TB with additional resistance to quinolones and second-line injectable agents).

Cervical epithelia have numerous functions that include proliferation, differentiation, maintenance of fluid balance, protection from environmental hazards, and paracellular transport of solutes via tight junctions (TJs). Epithelial functions must be tightly regulated during pregnancy and parturition as the cervix undergoes extensive growth and remodeling. This study evaluated TJ proteins, as well as markers of epithelial cell differentiation in normal and cervical ripening defective mice to gain insights into how the permeability barrier is regulated during pregnancy and parturition. Although numerous TJ proteins are expressed in the nonpregnant cervix, claudins 1 and 2 are temporally regulated in pregnancy. Claudin 1 mRNA expression is increased, whereas claudin 2 expression declines. The cellular localization of claudin 1 shifts at the end of pregnancy (gestation d 18.75) to the plasma membrane in a lattice pattern, consistent with TJs in the apical cells. The timing of claudin 1-enriched TJs coincides with initiation of terminal differentiation of cervical squamous epithelia as evidenced by the increased expression of genes by differentiated epithelia late on gestation d 18. The cervical ripening defective steroid 5alpha-reductase type 1 deficient mouse, which has an elevated local progesterone concentration, also has aberrant claudin 1 and 2 expressions, fails to form claudin 1-enriched TJs, and lacks normal expression of genes involved in epithelial terminal differentiation. These data suggest that changes in permeability barrier properties during cervical ripening are, in part, negatively regulated by progesterone, and that dynamic changes in barrier properties of the cervix occur during pregnancy and parturition.

T cell activation is driven by the TCR and complemented by costimulation. We have studied the dynamics of ligand-engagement of the costimulatory receptor CD2 in T cell/APC couples. Thousands of ligand-engaged CD2 molecules were included in a large T cell invagination at the center of the cellular interface within 1 min of cell couple formation. The structure and regulation of this invagination shared numerous features with phagocytosis and macropinocytosis. Three observations further characterize the invagination and the inclusion of CD2: 1) numerous ligand-engaged receptors were enriched in and internalized through the T cell invagination, none as prominently as CD2; 2) dissolution of the T cell invagination and CD2 engagement were required for effective proximal T cell signaling; and 3) the T cell invagination was uniquely sensitive to the affinity of the TCR for peptide-MHC. Based on this characterization, we speculate that the T cell invagination, aided by CD2 enrichment, internalizes parts of the TCR signaling machinery to reset T cell signaling upon agonist-mediated, stable APC contact.

Although peroxisomes oxidize lipids, the metabolism of lipid bodies and peroxisomes is thought to be largely uncoupled from one another. In this study, using oleic acid-cultured Saccharomyces cerevisiae as a model system, we provide evidence that lipid bodies and peroxisomes have a close physiological relationship. Peroxisomes adhere stably to lipid bodies, and they can even extend processes into lipid body cores. Biochemical experiments and proteomic analysis of the purified lipid bodies suggest that these processes are limited to enzymes of fatty acid beta oxidation. Peroxisomes that are unable to oxidize fatty acids promote novel structures within lipid bodies ("gnarls"), which may be organized arrays of accumulated free fatty acids. However, gnarls are suppressed, and fatty acids are not accumulated in the absence of peroxisomal membranes. Our results suggest that the extensive physical contact between peroxisomes and lipid bodies promotes the coupling of lipolysis within lipid bodies with peroxisomal fatty acid oxidation.

The Australian Mycobacterium Reference Laboratory Network collected and analysed laboratory data on new cases of disease caused by Mycobacterium tuberculosis complex in the year 2004. A total of 787 cases were identified by bacteriology, representing an annual reporting rate of 3.9 cases per 100,000 population. Almost all isolates were identified as M. tuberculosis (n = 785), the remaining isolates being one each of Mycobacterium africanum and Mycobacterium canettii. Seven children under 10 years of age (female n = 5, male n = 2) had bacteriologically confirmed tuberculosis (gastric aspirate n = 4, lymph node n = 1, pleural n = 1, thigh wound n = 1). Results of in vitro drug susceptibility testing were available for all 787 isolates for isoniazid (H), rifampicin (R), ethambutol (E), and pyrazinamide (Z). A total of 71 (9.0%) isolates of M. tuberculosis were resistant to at least one of these anti-tuberculosis agents. Resistance to at least both H and R (defined as multidrug resistance) was detected in 12 (1.5%) isolates; 10 were from the respiratory tract (sputum n = 7, bronchoscopy n = 3). The country of birth was known for 68/71 (95.8%) cases with a drug resistant strain; eight were Australian, 60 were overseas born, and three were unknown. Of the 60 migrants with drug resistant disease, 37 (61.7%) were from three countries; Viet Nam (n = 20), China (n = 9) and India (n = 8).

In 2003, a steer carcass was condemned at a Central Queensland abattoir because of metastatic tumors. In addition, a granulomatous lesion was found in the mediastinal lymph node. Histological examination showed this to be a pyogranuloma, typically associated with Rhodococcus or the Nocardia/Streptomyces group. However, in this case, the only etiological agent was an acid-fast bacillus, which would normally be associated with a more fibrous lesion. A number of nucleic acid-based techniques were used, and the isolate was identified as Mycobacterium asiaticum. This organism is a rarely encountered opportunistic pathogen of humans, associated with subtropical climates. This is the first report of this organism causing infection in cattle. The similarities between this case and cases of human disease are discussed.

The Australian Mycobacterium Reference Laboratory Network collected and analysed laboratory data on new cases of disease caused by Mycobacterium tuberculosis complex in the year 2003. A total of 784 cases were identified by bacteriology, representing an annual reporting rate of 3.9 cases of laboratory confirmed tuberculosis per 100,000 population. The most commonly encountered culture-positive specimens were sputum (n = 351), lymph node (n = 176) and from bronchoscopy (n = 97). Smears containing acid fast bacilli were present in sputum (53.0%), bronchoscopy (32.0%) and lymph node (23.3%). Five children (female n = 3, male n = 2) under 10 years of age had bacteriologically confirmed tuberculosis. Eighty isolates of M. tuberculosis and one of Mycobacterium africanum (10.3%) were resistant to at least one of the standard anti-tuberculosis agents. Mono-resistance to isoniazid, ethambutol, rifampicin, and pyrazinamide was detected in 45, three, two, and one isolates respectively. Multidrug-resistance (MDRTB) defined as resistance to both isoniazid and rifampcin was observed in seven (0.9%) isolates. Of the seven MDRTB isolates, six were from the respiratory tract and four were from smear positive specimens. Of the 81 patients with drug resistant isolates, 78 (96.3%) were classified as having initial resistance; two had acquired resistance and no information was available for one isolate; five were Australian-born; and 76 (93.8%) had migrated from a total of 30 countries.

Setting: New cases of pulmonary tuberculosis (TB) were noted in a cluster of young Caucasian males, an unusual ethnic group for this disease in Queensland, Australia. It was noted that marijuana water pipe ('bong') smoking was common amongst cases and contacts., Objective: To report this cluster of TB and to investigate whether shared use of a marijuana water pipe was associated with transmission of TB., Design: All contacts were identified and screened according to standard protocols. Cases were asked to list contacts with whom they had shared a marijuana water pipe., Results: Five cases of open pulmonary TB were identified clinically and on sputum culture, and all isolates of Mycobacterium tuberculosis were identical on typing. Of 149 contacts identified, 114 (77%) completed screening, and 57 (50%) had significant tuberculin skin test (TST) reactions on follow-up. Of 45 contacts who had shared a marijuana water pipe with a case, 29 (64%) had a significant TST reaction., Conclusion: Sharing a marijuana water pipe with a case of pulmonary TB was associated with transmission of TB (OR 2.22, 95 % CI 0.96-5.17), although the most important risk factor for acquiring TB infection in this cluster was close household contact with a case (OR 4.91, 95% CI 1.13-20.70).

The Australian Mycobacterium Reference Laboratory Network collected and analysed laboratory data on new cases of disease caused by Mycobacterium tuberculosis complex in the year 2001. A total of 771 cases were identified, representing an annual reporting rate of 4.0 cases of laboratory-confirmed tuberculosis per 100,000 population. The predominant specimen type was sputum, (n=369) and a further 111 were collected at bronchoscopy. Smears were positive for 214 of 369 (58.0%) sputum and 42 of 111 (37.8%) bronchoscopy specimens respectively. Seven children (male n=5, female n=2) under 10 years of age had bacteriologically confirmed tuberculosis. A total of 69 isolates (8.9%), comprising 67 M. tuberculosis, one M. africanum, and one M. bovis, were resistant to at least one of the anti-tuberculosis agents. Excluding the M. bovis isolate, 61 of 64 (93.5%) were classified as having initial resistance, three had acquired resistance, and no data were available on the presence or absence of previous treatment for four patients. Resistance to at least isoniazid and/or rifampicin was noted for 67 isolates (8.7%), with resistance to both isoniazid and rifampicin (i.e. defined as multidrug-resistant disease) observed in 12 (1.6%) isolates. All of the multidrug-resistant isolates were M. tuberculosis, 10 were from the respiratory tract. The country of birth was known for 63 of 68 (92.6%) patients with a drug-resistant strain of M. tuberculosis or M. africanum; five were Australian-born and 58 (92.1%) had migrated from a total of 22 countries. One hundred and seven respiratory specimens had a nucleic acid amplification testing performed; 89 of 90 (98.9%) smear positives were nucleic acid amplification testing positive, whilst only 13 of 17 (76.5%) smear negative specimens were nucleic acid amplification testing positive. The 2001 laboratory data reveals a stable incidence rate and level of drug resistance in isolates from Australian patients with tuberculosis.

The extracellular matrix is an intricate network of macromolecules which provides support for cells and a framework for tissues. The detailed structure and organisation of most matrix polymers is poorly understood. These polymers have a complex ultrastructure, and it has proved a major challenge both to define their structural organisation and to relate this to their biological function. However, new approaches using automated electron tomography are beginning to reveal important insights into the molecular assembly and structural organisation of two of the most abundant polymer systems in the extracellular matrix. We have generated three-dimensional reconstructions of collagen fibrils from bovine cornea and fibrillin microfibrils from ciliary zonules. Analysis of these data has provided new insights into the organisation and function of these large macromolecular assemblies.

The Australian Mycobacterium Reference Laboratory Network collected and analysed laboratory data on new diagnoses of disease caused by Mycobacterium tuberculosis complex in the year 2000. A total of 765 cases were identified, representing an annual reporting rate of 4.0 cases of laboratory-confirmed tuberculosis (TB) per 100,000 population. Pulmonary disease was diagnosed in 64.9 per cent of cases with a male:female ratio of 1.5:1. Smears were positive for 209/365 (57.3%) of sputum isolates and 39/117 (33.3%) bronchoscopy isolates. Sputum from males was more likely to be smear-positive (63.3%) than from females (47.5%). Isolates from lymph node accounted for 136 (17.7%) of all cases; only 28.7 per cent were smear-positive. Eighty-four (11.0%) isolates, comprising 82 M. tuberculosis and 2 M. bovis strains, demonstrated in vitro resistance to at least one of the standard anti-TB medications. Resistance to at least isoniazid and rifampicin (defined as multidrug-resistant TB) was observed for only 8 (1.0%) strains, a rate similar to previous years. Almost all (96.3%) of patients with drug resistant strains were classified as having initial resistance. The country of birth was known for 76 (92.7%) of 82 patients with a drug resistant strain of M. tuberculosis; 6 were Australian-born and 70 (92.1%) had migrated from a total of 17 countries. Of these 70 migrants with drug-resistant disease, 68.6 per cent had migrated from one of the following countries: Vietnam (n=15), China (n=11), Philippines (n=11), India (n=6), and Indonesia (n=5).

The ability of the cornea to transmit light while being mechanically resilient is directly attributable to the formation of an extracellular matrix containing orthogonal sheets of collagen fibrils. The detailed structure of the fibrils and how this structure underpins the mechanical properties and organization of the cornea is understood poorly. In this study, we used automated electron tomography to study the three-dimensional organization of molecules in corneal collagen fibrils. The reconstructions show that the collagen molecules in the 36-nm diameter collagen fibrils are organized into microfibrils (approximately 4-nm diameter) that are tilted by approximately 15 degrees to the fibril long axis in a right-handed helix. An unexpected finding was that the microfibrils exhibit a constant-tilt angle independent of radial position within the fibril. This feature suggests that microfibrils in concentric layers are not always parallel to each other and cannot retain the same neighbors between layers. Analysis of the lateral structure shows that the microfibrils exhibit regions of order and disorder within the 67-nm axial repeat of collagen fibrils. Furthermore, the microfibrils are ordered at three specific regions of the axial repeat of collagen fibrils that correspond to the N- and C-telopeptides and the d-band of the gap zone. The reconstructions also show macromolecules binding to the fibril surface at sites that correspond precisely to where the microfibrils are most orderly.

The medical community and general population have become aware that genetic testing is available to look for BRCA1 and BRCA2 mutations. However, criteria for who should be referred for genetic counseling and possible subsequent testing have yet to be determined, and many genetics centers have been overwhelmed by the demand for service. We set out to develop a family history assessment tool (FHAT) that could be used by physicians to select individuals for genetic counseling. Arbitrarily, we chose individuals who would have an approximate doubling of their lifetime risk for breast or ovarian cancer. The FHAT was then applied to 184 unrelated families, with an index patient who had breast or ovarian cancer and who had accepted the offer of BRCA1 BRCA2 testing. Data were compiled to compare the number of individuals who would have been referred for genetic counseling and the number of mutation-positive individuals who would have been screened out from counseling using FHAT, the tables from Claus, and the BRCAPRO system. In this population, FHAT was effective in minimizing both the number of referrals and the likelihood of missing women who were later found to be mutation-positive.