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In 2018, the Ministry of the Environment and Water, with the support of the German International Cooperation Agency (Gesellschaft für Internationale Zusammenarbeit GIZ), evaluated the possibility of developing a marine and coastal spatial planning process in the northern coastal area of Manabí province, Ecuador. Based on the outcomes from workshops and interviews in the territory, the main socio-economic and environmental issues related to management in the coastal zone were identified. The uses resources in the area (artisanal fishing, conservation, industrial fishing and tourism), and the conflicts between users/ stakeholders were determined. The local communities expressed their willingness to initiate a marine and coastal management and ordering process due to their perception indicating that the rules are not being met and the existence of several conflicts, mainly between industrial and small-scale (artisanal) fishing. From the beginning of the project, we worked together with the local coastal communities, and the application of spatial analysis tools and remote sensors that served to alleviate the scarcity of available information. A methodology was then presented to assess the pre-feasibility of the area, with the aim to define how feasible, it is to carry out the process of planning marine and coastal uses. Once the viability was analyzed, three future management scenarios are proposed along with recommendations for the management of the area and the lessons learned. The study area has a high pre-feasibility to begin a process de marine and coastal planning due to the support of the local communities and the limited conflicts. Thus, it is recommended that this type of process should be promoted by the environmental authority by engaging and empowering resource users, before the conflicts are exacerbated and the communities and local governments lose interest due to lack of institutional support.

BackgroundThe MODS is an important assay for early diagnosis of tuberculosis and drug susceptibility. MODS is based in the microscopic observation, underneath, of the characteristic cords of Mycobacterium tuberculosis colonies grown in liquid media. An inverted optical microscope is required to observe and interpret MODS cultures. Unfortunately, the cost of commercial inverted microscopes is not affordable in low resource settings in developing countries.MethodologyTo perform a diagnosis of tuberculosis using the MODS assay, images with modest quality are enough for proper interpretation. Therefore, the use of a high cost commercial inverted optical microscope is not indispensable. In this study, we designed a prototype of an optical inverted microscope created with a 3D printer and based on a smartphone. The system was evaluated by comparison of manual interpretations of 226 TB positive MODS culture images and 207 negative MODS culture images.SignificanceThe prototype resulted in a low-cost inverted optical microscope, with simple functioning, and whose parts have been manufactured using 3D printing techniques. The quality of the images was good enough and achieved a 100% concordance between the manual inspection with the developed microscope, and the standard diagnostics of MODS.

Tuberculosis (TB) control efforts are hampered by a mismatch in diagnostic technology. Lack of adequate early diagnostics and Multi-drug resistant (MDR) detection is a critical problem in control efforts. Alternate and novel diagnostic approaches are required, especially in low-resources settings where they are needed most. The Microscopic Observation Drug Susceptibility (MODS) assay is a cost-effective, highly sensitive, and specific method based on the detection of characteristic cording growth patterns of Mycobacterium tuberculosis (MTB), in microscopic examination of a liquid culture under an inverted microscope. By adding antimicrobials to the wells, MODS also determines antimicrobial susceptibility in both MDR and Extreme Drug Resistant (XDR) tuberculosis. The interpretation of a MODS culture performed in a 24 well plate, requires an extensive inspection over the entire surface to detect TB cords. This process requires significant time and effort from a trained microscopist. We evaluated a lens-free imager system, able to render microscopic images of live specimens, for the proof of principle to be used for MODS culture interpretation. The lens-free imager system is able to digitalize a 24-mm(2) surface with approximately 40X magnification in a single capture. The evaluation of the lens-free imager found that it produced microscopic images that were adequate for MODS interpretation by a human expert. Compared to the average time that takes a microscopist to completely examine a MODS culture sample, the lens free imager notably reduced the time of inspection. Therefore, lens-free imager variants may constitute promising systems to aid in the diagnostics of tuberculosis, by simplifying and reducing the time of inspection and permitting automatization of MODS interpretation.

Cough is a characteristic symptom of tuberculosis, is the main cause of transmission, and is used to assess treatment response. We aimed to identify the best measure of cough severity and characterize changes during initial tuberculosis therapy. We conducted a prospective cohort of recently diagnosed ambulatory adult patients with pulmonary tuberculosis in two tertiary hospitals in Lima, Peru. Pre-treatment and five times during the first two months of treatment, a vibrometer was used to capture 4-hour recordings of involuntary cough. A total of 358 recordings from 69 participants were analyzed using a computer algorithm. Total time spent coughing (seconds per hour) was a better predictor of microbiologic indicators of disease severity and treatment response than the frequency of cough episodes or cough power. Patients with prior tuberculosis tended to cough more than patients without prior tuberculosis, and patients with tuberculosis and diabetes coughed more than patients without diabetes co-morbidity. Cough characteristics were similar regardless of HIV co-infection and for drug-susceptible versus drug-resistant tuberculosis. Tuberculosis treatment response may be meaningfully assessed by objectively monitoring the time spent coughing. This measure demonstrated that cough was increased in patients with TB recurrence or co-morbid diabetes, but not because of drug resistance or HIV co-infection.

Background Congregate settings may serve as institutional amplifiers of tuberculosis (TB) and multidrug-resistant tuberculosis (MDR-TB). We analyze spatial, epidemiological, and pathogen genetic data prospectively collected from neighborhoods surrounding a prison in Lima, Peru, where inmates experience a high risk of MDR-TB, to investigate the risk of spillover into the surrounding community. Methods Using hierarchical Bayesian statistical modeling, we address three questions regarding the MDR-TB risk: (i) Does the excess risk observed among prisoners also extend outside the prison? (ii) If so, what is the magnitude, shape, and spatial range of this spillover effect? (iii) Is there evidence of additional transmission across the region? Results The region of spillover risk extends for 5.47 km outside of the prison (95% credible interval: 1.38, 9.63 km). Within this spillover region, we find that nine of the 467 non-inmate patients (35 with MDR-TB) have MDR-TB strains that are genetic matches to strains collected from current inmates with MDR-TB, compared to seven out of 1080 patients (89 with MDR-TB) outside the spillover region (p values: 0.022 and 0.008). We also identify eight spatially aggregated genetic clusters of MDR-TB, four within the spillover region, consistent with local transmission among individuals living close to the prison. Conclusions We demonstrate a clear prison spillover effect in this population, which suggests that interventions in the prison may have benefits that extend to the surrounding community. Electronic supplementary material The online version of this article (10.1186/s12916-018-1111-x) contains supplementary material, which is available to authorized users.

To evaluate the performance of diagnostic tools for diabetes mellitus, including laboratory methods and clinical risk scores, in newly-diagnosed pulmonary tuberculosis patients from four middle-income countries.In a multicentre, prospective study, we recruited 2185 patients with pulmonary tuberculosis from sites in Indonesia, Peru, Romania and South Africa from January 2014 to September 2016. Using laboratory-measured glycated haemoglobin (HbA1c) as the gold standard, we measured the diagnostic accuracy of random plasma glucose, point-of-care HbA1c, fasting blood glucose, urine dipstick, published and newly derived diabetes mellitus risk scores and anthropometric measurements. We also analysed combinations of tests, including a two-step test using point-of-care HbA1cwhen initial random plasma glucose was ≥ 6.1 mmol/L.The overall crude prevalence of diabetes mellitus among newly diagnosed tuberculosis patients was 283/2185 (13.0%; 95% confidence interval, CI: 11.6-14.4). The marker with the best diagnostic accuracy was point-of-care HbA1c (area under receiver operating characteristic curve: 0.81; 95% CI: 0.75-0.86). A risk score derived using age, point-of-care HbA1c and random plasma glucose had the best overall diagnostic accuracy (area under curve: 0.85; 95% CI: 0.81-0.90). There was substantial heterogeneity between sites for all markers, but the two-step combination test performed well in Indonesia and Peru.Random plasma glucose followed by point-of-care HbA1c testing can accurately diagnose diabetes in tuberculosis patients, particularly those with substantial hyperglycaemia, while reducing the need for more expensive point-of-care HbA1c testing. Risk scores with or without biochemical data may be useful but require validation.Évaluer la performance des outils de diagnostic du diabète sucré, et notamment les méthodes de laboratoire et les cotes de risque clinique, chez les patients souffrant d'une tuberculose pulmonaire récemment diagnostiquée dans quatre pays à revenu intermédiaire.Dans le cadre d'une étude multicentrique et prospective, nous avons recruté 2185 patients atteints de tuberculose pulmonaire dans différents endroits en Afrique du Sud, en Indonésie, au Pérou et en Roumanie entre janvier 2014 et septembre 2016. En utilisant comme référence l'hémoglobine glyquée (HbA1c) mesurée en laboratoire, nous avons évalué la précision diagnostique de la glycémie aléatoire, de l'HbA1c sur le lieu d'intervention, de la glycémie à jeun, de la bandelette urinaire, des cotes de risque déjà publiées et récemment obtenues pour le diabète sucré et des mesures anthropométriques. Nous avons également analysé des combinaisons de tests, et notamment un test en deux étapes qui utilise l'HbA1c sur le lieu d'intervention lorsque la glycémie aléatoire initiale est ≥ 6,1 mmol/L.La prévalence brute générale du diabète sucré chez les patients souffrant d'une tuberculose récemment diagnostiquée était de 283/2185 (13,0%; intervalle de confiance, IC, à 95%: 11,6–14,4). Le marqueur fournissant la meilleure précision diagnostique était l'HbA1c sur le lieu d'intervention (aire sous la courbe ROC: 0,81; IC à 95%: 0,75–0,86). La meilleure précision diagnostique générale a été obtenue à l'aide d'une cote de risque calculée au moyen de l'âge, de l'HbA1c sur le lieu d'intervention et de la glycémie aléatoire (aire sous la courbe ROC: 0,85; IC à 95%: 0,81–0,90). Une forte hétérogénéité entre les différents endroits a été constatée pour tous les marqueurs, mais le test en deux étapes a donné de bons résultats en Indonésie et au Pérou.L'utilisation de la glycémie aléatoire suivie de l'HbA1c sur le lieu d'intervention permet de diagnostiquer avec précision le diabète chez les patients atteints de tuberculose, en particulier lorsqu'ils souffrent d'une importante hyperglycémie, tout en diminuant le besoin de tests HbA1c sur le lieu d'intervention, plus coûteux. Les cotes de risque associées ou non à des données biochimiques peuvent être utiles, mais doivent être validées.Evaluar el rendimiento de las herramientas de diagnóstico para la diabetes mellitus, incluidos los métodos de laboratorio y los índices de riesgo clínico, en pacientes recién diagnosticados con tuberculosis pulmonar de cuatro países de ingresos medios.En un estudio prospectivo multicéntrico, se seleccionaron 2185 pacientes con tuberculosis pulmonar de algunas regiones de Indonesia, Perú, Rumanía y Sudáfrica entre enero de 2014 y septiembre de 2016. Usando hemoglobina glicosilada medida en laboratorio (HbA1c) como el patrón de excelencia, se midió la precisión diagnóstica de glucosa plasmática al azar, estando HbA1c en el punto de atención, glucemia en sangre en ayunas, tira reactiva de orina, índices de riesgo de diabetes mellitus publicados recientemente y medidas antropométricas. También se analizaron combinaciones de pruebas, incluida una prueba de dos pasos con HbA1c en el punto de atención cuando la glucosa plasmática aleatoria inicial fue ≥ 6,1mmol/L.La prevalencia general cruda de diabetes mellitus entre los pacientes con tuberculosis recién diagnosticados fue de 283/2185 (13,0 %, intervalo de confianza del 95 %, IC: 11,6–14,4). El marcador con la mejor precisión diagnóstica fue la HbA1c en el punto de atención (área bajo la curva característica operativa del receptor: 0,81; IC del 95 %: 0,75–0,86). Un índice de riesgo derivado de la edad, la HbA1c en el punto de atención y la glucosa plasmática al azar obtuvieron la mejor precisión diagnóstica general (área bajo la curva: 0,85; IC del 95 %: 0,81–0,90). Hubo una heterogeneidad sustancial entre las regiones para todos los marcadores, pero la prueba combinada de dos pasos funcionó de manera correcta en Indonesia y Perú.La glucosa plasmática al azar seguida de la prueba de HbA1c en el punto de atención puede diagnosticar con precisión la diabetes en pacientes con tuberculosis, particularmente en aquellos con hiperglucemia sustancial, mientras se reduce la necesidad de pruebas más costosas de HbA1c en el punto de atención. Los índices de riesgo con o sin datos bioquímicos pueden ser útiles, pero requieren validación.تقييم أداء أدوات تشخيص مرض السكري، بما في ذلك الطرق المختبرية ودرجات المخاطر السريرية، لدى المرضى الذين تم تشخيص إصابتهم حديثاً بالسل الرئوي من أربعة بلدان متوسطة الدخل.في دراسة استباقية متعددة المراكز، قمنا بالاستعانة بـ 2185 مريضًا بمرض السل الرئوي من مواقع في إندونيسيا وبيرو وجنوب إفريقيا ورومانيا من يناير/كانون ثاني 2014 إلى سبتمبر/أيلول 2016. وعن طريق قياس الهيموغلوبين الغليكوزيلاتي في المختبر (HbA1c) كمعيار أساسي، قمنا بقياس مدى دقة تشخيص كل من جلوكوز البلازما العشوائي، واختبار HbA1c في أماكن الرعاية، وجلوكوز الدم أثناء الصيام، ومقياس البول، ودرجات المخاطر المنشورة والمستنبطة حديثا من داء السكري، والقياسات الأنثروبومترية. قمنا أيضا بتحليل مجموعات من الاختبارات، بما في ذلك اختبار من خطوتين باستخدام اختبار HbA1c في أماكن الرعاية عندما كان مستوى جلوكوز البلازما العشوائي أكبر من أو يساوي 6.1 مليمول/ لتر.كان معدل الانتشار الكلي لمرض السكري بين المرضى الذين تم تشخيص إصابتهم حديثًا بالسل هو 283/2185 (13.0%؛ بفاصل ثقة 95%، فاصل ثقة: 11.6 إلى 14.4).كانت العلامة ذات أفضل دقة تشخيصية هي اختبار HbA1c في أماكن الرعاية (المساحة تحت منحنى خاصية التشغيل للمستقبِل: 0.81؛ بفاصل ثقة 95%: 0.75 إلى 0.86). كانت درجة الخطر المشتقة باستخدام العمر، واختبار HbA1c في أماكن الرعاية، وجلوكوز البلازما العشوائي، هي أفضل دقة تشخيصية عامة (المساحة تحت المنحنى: 0.85؛ بفاصل ثقة 95%: 0.81 إلى 0.90). كانت هناك عدم تجانس واضح بين المواقع لكل العلامات، إلا أن الاختبار المكون من خطوتين قدم أداءً جيدًا في إندونيسيا وبيرو.يمكن لاختبار جلوكوز البلازما العشوائي، ويليه اختبار HbA1c في أماكن الرعاية، تشخيص مرض السكري بدقة في مرضى السل، وخاصة أولئك الذين يعانون من ارتفاع شديد في سكر الدم، مع تقليل الحاجة إلى اختبار HbA1c الأكثر تكلفة في أماكن الرعاية. قد تكون درجات الخطر مفيدة، سواء مع بيانات كيميائية حيوية أو بدونها، ولكنها تتطلب التحقق من الصحة.评估用于四个中等收入国家新诊断结核病患者的糖尿病诊断工具的表现，其中包括实验室方法和临床风险评分。.在这项多中心、前瞻性的研究中，自 2014 年 1 月起至 2016 年 9 月，我们从秘鲁、罗马尼亚、南非和印度尼西亚各地招募了 2185 名肺结核患者。使用实验室测量的糖化血红蛋白 (HbA1c) 为黄金标准，我们测量了随机血糖、糖化血红蛋白 (HbA1c) 床旁检测、空腹血糖、尿液常规检查、已发表的和新导出的糖尿病风险评分以及人体测量法的诊断准确度。我们还对组合检测法进行了分析，其中包括两步检测法：即在初始随机血糖为临界值 ≥6.1 mmol/L 时，进行糖化血红蛋白 (HbA1c) 床旁检测。.新诊断结核病患者中，糖尿病的整体患病率为 283/2185（13.0％；95％ 置信区间，CI：11.6–14.4）。最准确的诊断指标是糖化血红蛋白 (HbA1c) 床旁检测（受试者工作特征曲线下的区域：0.81；95％ 置信区间，CI：0.75-0.86）。使用年龄、糖化血红蛋白 (HbA1c) 床旁检测和随机血糖得出的风险评分整体诊断准确度最佳（受试者工作特征曲线下的区域：0.85；95％ 置信区间，CI：0.81-0.90）。不同地点的指标之间存在显著的异质性，但两步组合检测法在秘鲁和印度尼西亚表现良好。.在随机血糖检测后再进行糖化血红蛋白 (HbA1c) 床旁检测可以准确诊断结核病患者是否患有糖尿病（尤其针对患有严重高血糖的患者），同时减少对更为昂贵的糖化血红蛋白 (HbA1c) 床旁检测的需求。内附或未附生化指标的风险评分可能有用，但仍需验证。.Оценить эффективность инструментов диагностики сахарного диабета (включая лабораторные методы и показатели клинического риска) у пациентов с недавно диагностированным туберкулезом легких из четырех стран со средним уровнем доходов.С января 2014 года по сентябрь 2016 года авторы провели многоцентровое проспективное исследование, в котором приняли участие 2185 пациентов с туберкулезом легких, проживающих в Индонезии, Перу, Румынии и Южной Африке. Используя измеренный в лаборатории гликозилированный гемоглобин (HbA1c) в качестве общепринятого стандарта, авторы измерили диагностическую точность выборочного определения уровня глюкозы в плазме; уровня HbA1c, определяемого в месте предоставления медицинской помощи; уровня глюкозы в крови натощак и определения сахара в моче с помощью индикаторной полоски с учетом опубликованных и недавно полученных оценок риска заболевания сахарным диабетом и антропометрических измерений. Мы также проанализировали комбинации тестов, в том числе двухэтапный тест с использованием HbA1c, измеренного в месте предоставления медицинской помощи, при начальном случайном уровне глюкозы в плазме ≥ 6,1 ммоль/л.Общая распространенность сахарного диабета среди пациентов с недавно выявленным туберкулезом составила 283 случая на 2185 человек (13,0%, 95%-й доверительный интервал, ДИ: 11,6–14,4). Маркер с наибольшей диагностической точностью — HbA1c, измеренный в месте предоставления медицинской помощи (область под кривой ROC: 0,81; 95%-й ДИ: 0,75–0,86). Оценка риска в зависимости от возраста, HbA1c, измеренного в месте предоставления медицинской помощи, и выборочного определения глюкозы в плазме имела наилучшую общую диагностическую точность (область под кривой: 0,85; 95%-й ДИ: 0,81–0,90). В различных местах наблюдалась существенная гетерогенность всех маркеров, при этом двухступенчатый комбинированный тест дал хорошие показатели для Индонезии и Перу.Выборочно определяемый уровень глюкозы в плазме с последующим тестированием HbA1c в месте предоставления медицинской помощи может точно диагностировать диабет у пациентов с туберкулезом, особенно у пациентов с существенной гипергликемией, уменьшая необходимость в более дорогостоящем тестировании HbA1c в месте предоставления медицинской помощи. Оценки риска по данным биохимического анализа крови или без них могут быть полезными, но нуждаются в проверке.

Barley (Hordeum vulgare L.) is produced in the Ecuadorian highlands ( > 3,000 m a.s.l.) primarily for self-consumption and small-scale commercialization. Not many crop species are adapted to this altitude; therefore, barley is one of a few crop species that can be grown at these locations. Severe environmental conditions can be found in the Ecuadorian highlands since the region is characterized by poor soils and water deficiency (< 300 mm yr-1). The Instituto Nacional de Investigaciones Agropecuarias (INIAP) has developed 'INIAP-Palmira 2014', a hulled two-row barley variety adapted to Ecuadorian agricultural conditions in the highlands. 'INIAP-Palmira 2014' showed acceptable yield performance as compared with the most popular improved barley cultivars in different production areas located in Ecuador. However, this new barley variety showed superior performance under water stress conditions in the highlands highlands (>3,000 m a.s.l.). Additionally, 'INIAP-Palmira 2014' showed disease resistance, mainly to yellow rust, in all of the locations where the new variety was evaluated.

The presence of a cavitary lesion is a radiographic hallmark of pulmonary tuberculosis (TB). The sizes of the cavitary lesion and its proximity to the bronchial tree have been associated with mycobacterial burden. However, the task of evaluating the size of a cavitary lesion is problematic due to differences in reporting methods (e.g., uni-dimensional maximum in the axial plane vs. bi-dimensional) and inter-observer variance. The evaluation of a cavitary lesion is often complicated by their irregular shape, thus the radiographic reporting of the lesion (e.g., spherical volume) might underestimate or overestimate its actual size. The problem with having several methods of reporting is that global communication becomes impaired, and results from different groups more difficult to compare. A computer-aided algorithm would help to diminish the inter-rater differences in reporting, and provide a more precise evaluation of the size of a lesion and its distance from the bronchial tree...

Background: Direct sputum smear is still the first-choice tool for screening of tuberculosis worldwide. Variants of this technique, to improve the sensitivity are desired. Methods: Two microbiological variants of the standard sputum smear ("pellet" and "diluted-pellet") for both Ziehl-Neelsen (ZN) and auramine fluorescence (AF) staining were evaluated. In addition, two methods for concentration of mycobacteria in sputum, using positive and negative pressure filtration, were tested and compared. The evaluation of the microbiological variants was performed on 98 culture positive sputum samples from different TB patients. The diagnostics sensitivity and the level of detritus in the processed sputum were determined. Bacilli load in the smear variants was determined by microscopic observation and by manual inspection of microscopic digital images. The comparison of the mycobacteria filtration methods was performed on 76 smear positive sputum samples. Filters retaining the concentrated mycobacteria were stained with AF and compared with the direct smear. Bacilli load, detritus level, filtered volume, filtration time and background noise level, were determined. Results: The sensitivity of microscopy with the microbiological variants was 7.1% and 2% higher in ZN and AF respectively, compared to direct smear. The sensitivity of AF in diluted pellet was significantly higher than all ZN variants (P < 0.05). Detritus level observed in slides was significantly lower in the diluted pellet than the pellet and direct smear in ZN and AF (P < 0.001). A significant increase in the bacilli load in microscopic observation and digital images analysis was observed in pellet and diluted pellet than the direct method (P

Hospital infection control measures are crucial to tuberculosis (TB) control strategies within settings caring for human immunodeficiency virus (HIV)-positive patients, as these patients are at heightened risk of developing TB. Pyrazinamide (PZA) is a potent drug that effectively sterilizes persistent Mycobacterium tuberculosis bacilli. However, PZA resistance associated with mutations in the nicotinamidase/pyrazinamidase coding gene, pncA, is increasing. A total of 794 patient isolates obtained from four sites in Lima, Peru, underwent spoligotyping and drug resistance testing. In one of these sites, the HIV unit of Hospital Dos de Mayo (HDM), an isolation ward for HIV/TB coinfected patients opened during the study as an infection control intervention: circulating genotypes and drug resistance pre- and postintervention were compared. All other sites cared for HIV-negative outpatients: genotypes and drug resistance rates from these sites were compared with those from HDM. HDM patients showed high concordance between multidrug resistance, PZA resistance according to the Wayne method, the two most common genotypes (spoligotype international type [SIT] 42 of the Latino American-Mediterranean (LAM)-9 clade and SIT 53 of the T1 clade), and the two most common pncA mutations (G145A and A403C). These associations were absent among community isolates. The infection control intervention was associated with 58-92% reductions in TB caused by SIT 42 or SIT 53 genotypes (odds ratio [OR] = 0.420, P = 0.003); multidrug-resistant TB (OR = 0.349, P < 0.001); and PZA-resistant TB (OR = 0.076, P < 0.001). In conclusion, pncA mutation typing, with resistance testing and spoligotyping, was useful in identifying a nosocomial TB outbreak and demonstrating its resolution after implementation of infection control measures.

OBJECTIVES: In a pilot implementation project of the microscopic-observation drug-susceptibility methodology, we conducted a process evaluation to identify health system and logistic challenges that need to be addressed in order to harness the benefits of rolling out promising new diagnostic tools for multidrug-resistant tuberculosis (MDRTB). METHODS: Regional data relating to health system practices and performance related to the MDRTB diagnostic algorithm were collected at health center, local, and regional laboratories. RESULTS: Parallel implementation of a new test and an existing method creates demands on funds, personnel, sample transport, and information systems in addition to new test startup costs. Obviating the need for primary culture at intermediate laboratories through direct drug susceptibility testing (DST) at the regional reference laboratory significantly reduces delay. Field application of well-defined national guidelines for DST is patchy. If fidelity to national guidelines were perfect, DST requests would increase more than 50-fold, with important implications for laboratory capacity. CONCLUSIONS: Implementing a new MDRTB diagnostic presents challenges to the laboratory environment, the existing DST process, and the application of national guidelines in peripheral clinics. Assessing each element can maximize efficient use of a new tool. Specifically, strengthening systems for transferring samples to the laboratory and delivering results to the requesting clinic in addition to investing in personnel and laboratory resources are integral to harnessing the benefits of high-performance new diagnostic tests and can bring added value to other programs in the health care system.

The intersample and intrasample variability of the results obtained with the microplate Alamar blue assay for the indirect drug susceptibility testing of Mycobacterium tuberculosis was investigated. Between 1.2 and 8.5% of paired MICs differed by more than one twofold dilution, resulting in discordant susceptible-resistant designations at frequencies between 0.6% (rifampin) and 18.9% (ethambutol).

Summary Background Pyrazinamide (PZA) is the most important drug against the latent stage of tuberculosis (TB) and is used in both first and second line treatment regimens. The continued increase in multi-drug resistant TB and the prevalence of PZA resistance makes the development of alternative assays for prompt identification of PZA resistance all the more important. Methods We standardized and evaluated a quantitative variant of the Wayne assay (QW) for determining PZA resistance in Mycobacterium tuberculosis strains. This assay quantifies M. tuberculosis metabolism of PZA and production of pyrazinoic acid (POA) using visible spectrophotometry. We evaluated this method using PZA concentrations of 400 μg/ml and 800 μg/ml at incubation periods of 3, 5 and 7 days. M. tuberculosis strains from 68 sputum samples were also tested with the standard Wayne assay, Tetrazolium Microplate Assay (TEMA), Bactec 460TB and pncA sequencing. We compared QW and standard Wayne assay against a dichotomous reference classification using concordant Bactec 460TB and pncA sequencing. Secondarily, we determined the quantitative correlation between both QW values and TEMA's minimum inhibitory concentration (MIC) against Bactec 460TB percentage growth. Results The standard Wayne showed sensitivity of 88% and specificity of 97.5%, giving a Youden Index (YI) of 0.855 against reference tests. The QW showed maximum YI of 0.934 on day 7 at 400 μg/ml PZA with 96% sensitivity and 97.4% specificity. Absorbance OD values for 400 μg/ml PZA were more accurate than 800 μg/ml PZA. Although QW showed high accuracy for PZA susceptibility, it did not correlate quantitatively with Bactec percentage growth. TEMA testing was unreliable and did not correlate with Bactec results. Conclusions The proposed QW assay is an inexpensive method capable of providing standardization and automation of colorimetric PZA resistance testing, with better discriminatory than the standard Wayne assay.

One obstacle to wider use of rapid liquid culture-based tuberculosis diagnostics such as the microscopic observation drug susceptibility (MODS) assay is concern about cross-contamination. We investigated the rate of laboratory cross-contamination in MODS, automated MBBacT, and Lowenstein–Jensen (LJ) cultures performed in parallel, through triangulation of microbiologic (reculturing stored samples), molecular (spoligotype/RFLP), and clinical epidemiologic data. At least 1 culture was positive for Mycobacterium tuberculosis for 362 (11%) of 3416 samples; 53 were regarded as potential cross-contamination suspects. Cross-contamination accounted for 17 false-positive cultures from 14 samples representing 0.41% (14/3416) and 0.17% (17/10 248) of samples and cultures, respectively. Positive predictive values for MODS, MBBacT (bioMérieux, Durham, NC), and LJ were 99.1%, 98.7%, and 99.7%, and specificity was 99.9% for all 3. Low rates of cross-contamination are achievable in mycobacterial laboratories in resource-poor settings even when a large proportion of samples are infectious and highly sensitive liquid culture-based diagnostics such as MODS are used.

Objetivos. Implementar un sistema para el diagnóstico remoto de tuberculosis y multidrogorresistencia (MDR) usando el método Microscopic-Observation Drug Susceptibility Assay (MODS) en el Laboratorio de Micobacterias del Centro de Excelencia en Tuberculosis de Trujillo (CENEX-Trujillo). El sistema incluyó una variante de un algoritmo de reconocimiento de Mycobacterium tuberculosis recientemente reportado a partir de imágenes digitales de cultivos MODS de muestras de esputo. Materiales y métodos. Se optimizó un algoritmo de reconocimiento por medio de un reentrenamiento del modelo estadístico basado en imágenes digitales de cultivos MODS provenientes del Laboratorio de Micobacterias del CENEX-Trujillo. Se obtuvieron imágenes de 50 cultivos MODS positivos de pacientes con sospecha de tuberculosis multidrogorresistente entre enero y octubre de 2012 en el CENEX-Trujillo. Resultados. La sensibilidad y la especificidad en objetos, para reconocer cordones de tuberculosis fueron de 92,04% y de 94,93% respectivamente. La sensibilidad y la especificidad en foto, para determinar un campo positivo a tuberculoisis fueron 95,4% y de 98,07% respectivamente. Conclusiones. Los resultados demostraron la factibilidad de la implementación de telediagnóstico en lugares remotos, el cual puede contribuir con la detección temprana de tuberculosis multidrogorresistente mediante el método MODS Objectives. To implement a system for remote diagnosis of tuberculosis and multidrug resistance (MDR) using the Microscopic-Observation Drug Susceptibility Assay (MODS) method in the Mycobacteria Laboratory, Trujillo Center of Excellence in Tuberculosis (CENEX-Trujillo). The system included a variant of an algorithm for recognition of Mycobacterium tuberculosis recently reported from digital images of MODS cultures of sputum samples. Materials and methods. The recognition algorithm was optimized using a retraining statistical model based on digital images of MODS cultures from CENEX-Trujillo. Images of 50 positive MODS cultures of patients with suspected multidrug-resistant tuberculosis were obtained between January and October 2012 in the CENEX-Trujillo. Results. The sensitivity and specificity to recognize strings of tuberculosis were 92.04% and 94.93% respectively using objects. The sensitivity and specificity to determine a positive tuberculosis field were 95.4% and 98.07% respectively using pictures. Conclusions. The results demonstrated the feasibility of the implementation of telediagnostics in remote locations, which may contribute to the early detection of multidrug-resistant tuberculosis by MODS method

Author(s): Trollip, AP; Moore, D; Coronel, J; Caviedes, L; Klages, S; Victor, T; Romancenco, E; Crudu, V; Ajbani, K; Vineet, VP; Rodrigues, C; Jackson, RL; Eisenach, K; Garfein, RS; Rodwell, TC; Desmond, E; Groessl, EJ; Ganiats, TG; Catanzaro, A | Abstract: ObjectiveTo establish breakpoint concentrations for the fluoroquinolones (moxifloxacin [MFX] and ofloxacin [OFX]) and injectable second-line drugs (amikacin [AMK], kanamycin [KM] and capreomycin [CPM]) using the microscopic observation drug susceptibility (MODS) assay.SettingA multinational study conducted between February 2011 and August 2012 in Peru, India, Moldova and South Africa.DesignIn the first phase, breakpoints for the fluoroquinolones and injectable second-line drugs (n = 58) were determined. In the second phase, MODS second-line drug susceptibility testing (DST) as an indirect test was compared to MGIT™ DST (n = 89). In the third (n = 30) and fourth (n = 156) phases, we determined the reproducibility and concordance of MODS second-line DST directly from sputum.ResultsBreakpoints for MFX (0.5 μg/ml), OFX (1 μg/ml), AMK (2 μg/ml), KM (5 μg/ml) and CPM (2.5 μg/ml) were determined. In all phases, MODS results were highly concordant with MGIT DST. The few discrepancies suggest that the MODS breakpoint concentrations for some drugs may be too low.ConclusionMODS second-line DST yielded comparable results to MGIT second-line DST, and is thus a promising alternative. Further studies are needed to confirm the accuracy of the drug breakpoints and the reliability of MODS second-line DST as a direct test.

BACKGROUND: Even though the WHO-endorsed, non-commercial MODS assay offers rapid, reliable TB liquid culture and phenotypic drug susceptibility testing (DST) at lower cost than any other diagnostic, uptake has been patchy. In part this reflects misperceptions about in-house assay quality assurance, but user convenience of one-stop procurement is also important. A commercial MODS kit was developed by Hardy Diagnostics (Santa Maria, CA, USA) with PATH (Seattle, WA, USA) to facilitate procurement, simplify procedures through readymade media, and enhance safety with a sealing silicone plate lid. Here we report the results from a large-scale field evaluation of the MODS kit in a government service laboratory. METHODS & FINDINGS: 2446 sputum samples were cultured in parallel in Lowenstein-Jensen (LJ), conventional MODS and in the MODS kit. MODS kit DST was compared with conventional MODS (direct) DST and proportion method (indirect) DST. 778 samples (31.8%) were Mycobacterium tuberculosis culture-positive. Compared to conventional MODS the sensitivity, specificity, positive, and negative predictive values (95% confidence intervals) of the MODS Kit were 99.3% (98.3-99.8%), 98.3% (97.5-98.8%), 95.8% (94.0-97.1%), and 99.7% (99.3-99.9%). Median (interquartile ranges) time to culture-positivity (and rifampicin and isoniazid DST) was 10 (9-13) days for conventional MODS and 8.5 (7-11) for MODS Kit (p

Tuberculosis control efforts are hampered by a mismatch in diagnostic technology: modern optimal diagnostic tests are least available in poor areas where they are needed most. Lack of adequate early diagnostics and MDR detection is a critical problem in control efforts. The Microscopic Observation Drug Susceptibility (MODS) assay uses visual recognition of cording patterns from Mycobacterium tuberculosis (MTB) to diagnose tuberculosis infection and drug susceptibility directly from a sputum sample in 7–10 days with a low cost. An important limitation that laboratories in the developing world face in MODS implementation is the presence of permanent technical staff with expertise in reading MODS. We developed a pattern recognition algorithm to automatically interpret MODS results from digital images. The algorithm using image processing, feature extraction and pattern recognition determined geometrical and illumination features used in an object-model and a photo-model to classify TB-positive images. 765 MODS digital photos were processed. The single-object model identified MTB (96.9% sensitivity and 96.3% specificity) and was able to discriminate non-tuberculous mycobacteria with a high specificity (97.1% M. avium, 99.1% M. chelonae, and 93.8% M. kansasii). The photo model identified TB-positive samples with 99.1% sensitivity and 99.7% specificity. This algorithm is a valuable tool that will enable automatic remote diagnosis using Internet or cellphone telephony. The use of this algorithm and its further implementation in a telediagnostics platform will contribute to both faster TB detection and MDR TB determination leading to an earlier initiation of appropriate treatment.

INTRODUCTION:Tuberculosis (TB) destroys lung tissues and this immunopathology is mediated in part by Matrix Metalloproteinases (MMPs). There are no data on the relationship between local tissue MMPs concentrations, anti-tuberculosis therapy and sputum conversion.MATERIALS AND METHODS:Induced sputum was collected from 68 TB patients and 69 controls in a cross-sectional study. MMPs concentrations were measured by Luminex array, TIMP concentrations by ELISA and were correlated with a disease severity score (TBscore). 46 TB patients were then studied longitudinally at the 2nd, 8th week and end of treatment.RESULTS:Sputum MMP-1,-2,-3,-8,-9 and TIMP-1 and -2 concentrations are increased in TB. Elevated MMP-1 and -3 concentrations are independently associated with higher TB severity scores (pCONCLUSIONS:MMPs are elevated in TB patients and associate with disease severity. This matrix-degrading phenotype resolves rapidly with treatment. The MMP profile at presentation correlates with a delayed treatment response

Multidrug-resistant tuberculosis (TB) threatens TB control worldwide. The microscopic observation drug susceptibility (MODS) assay is a low-cost, high-performance TB diagnostic tool for rapid liquid culture and direct isoniazid and rifampicin drug susceptibility testing (DST). The objective of this study was to explore the potential for extending the MODS assay to rapid second-line DST and to identify critical concentrations of candidate drugs for prospective testing. Sputum samples from 94 TB culture-positive patients receiving second-line TB agents were cultured following standardised MODS protocols, with a range of titrations of antimicrobial drugs added. Critical concentrations were determined using a modified Kaplan-Meier survival curve analysis. Candidate critical concentrations were determined for capreomycin (10 μg·mL(-1)), ciprofloxacin (1.25 μg·mL(-1)), cycloserine (40 μg·mL(-1)), ethambutol (10 μg·mL(-1)), ethionamide (5 μg·mL(-1)), kanamycin (5 μg·mL(-1)), para-aminosalicylic acid (10 μg·mL(-1)) and streptomycin (10 μg·mL(-1)). No cut-off point was identified for the other second-line drugs or for pyrazinamide. At particular concentrations of some second-line TB drugs this novel Kaplan-Meier analysis clearly differentiated populations that were susceptible or resistant. These candidate critical concentrations should now be tested in a range of epidemiological settings to define the performance of direct, second-line TB DST with MODS, offering potential low-cost second-line TB DST capacity.

Background Bleach-sedimentation may improve microscopy for diagnosing tuberculosis by sterilising sputum and concentrating Mycobacterium tuberculosis. We studied gravity bleach-sedimentation effects on safety, sensitivity, speed and reliability of smear-microscopy. Methods This blinded, controlled study used sputum specimens (n = 72) from tuberculosis patients. Bleach concentrations and exposure times required to sterilise sputum (n = 31) were determined. In the light of these results, the performance of 5 gravity bleach-sedimentation techniques that sterilise sputum specimens (n = 16) were compared. The best-performing of these bleach-sedimentation techniques involved adding 1 volume of 5% bleach to 1 volume of sputum, shaking for 10-minutes, diluting in 8 volumes distilled water and sedimenting overnight before microscopy. This technique was further evaluated by comparing numbers of visible acid-fast bacilli, slide-reading speed and reliability for triplicate smears before versus after bleach-sedimentation of sputum specimens (n = 25). Triplicate smears were made to increase precision and were stained using the Ziehl-Neelsen method. Results M. tuberculosis in sputum was successfully sterilised by adding equal volumes of 15% bleach for 1-minute, 6% for 5-minutes or 3% for 20-minutes. Bleach-sedimentation significantly decreased the number of acid-fast bacilli visualised compared with conventional smears (geometric mean of acid-fast bacilli per 100 microscopy fields 166, 95%CI 68-406, versus 346, 95%CI 139-862, respectively; p = 0.02). Bleach-sedimentation diluted paucibacillary specimens less than specimens with higher concentrations of visible acid-fast bacilli (p = 0.02). Smears made from bleach-sedimented sputum were read more rapidly than conventional smears (9.6 versus 11.2 minutes, respectively, p = 0.03). Counting conventional acid-fast bacilli had high reliability (inter-observer agreement, r = 0.991) that was significantly reduced (p = 0.03) by bleach-sedimentation (to r = 0.707) because occasional strongly positive bleach-sedimented smears were misread as negative. Conclusions Gravity bleach-sedimentation improved laboratory safety by sterilising sputum but decreased the concentration of acid-fast bacilli visible on microscopy, especially for sputum specimens containing high concentrations of M. tuberculosis. Bleach-sedimentation allowed examination of more of each specimen in the time available but decreased the inter-observer reliability with which slides were read. Thus bleach-sedimentation effects vary depending upon specimen characteristics and whether microscopy was done for a specified time, or until a specified number of microscopy fields had been read. These findings provide an explanation for the contradictory results of previous studies.

Background. Effective tuberculosis control is compromised by a lack of clarity about the timeframe of viable Mycobacterium tuberculosis shedding after treatment initiation under programmatic conditions. This study quantifies time to conversion from smear and culture positivity to negativity in unselected tuberculosis patients receiving standardized therapy in a directly observed therapy short-course (DOTS) program. Methods. Longitudinal cohort study following up 93 adults initiating tuberculosis therapy in Lima, Peru. Baseline culture and drug susceptibility tests (DSTs) were performed using the MBBacT, proportion, and microscopic observation drug susceptibility (MODS) methods. Smear microscopy and MODS liquid culture were performed at baseline and weekly for 4 weeks then every other week for 26 weeks. Results. Median conversion time from culture positivity to culture negativity of 38.5 days was unaffected by baseline smear status. Patients with fully susceptible tuberculosis had a median time to culture conversion of 37 days; 10% remained culture positive at day 60. Delayed culture conversion was associated with multidrug resistance, regardless of DST method used; non-multidrug resistance as defined by the proportion method and MODS (but not MBBacT) was also associated with delay. Persistent day 60 smear positivity yielded positive and negative predictive values of 67% and 92%, respectively, for detecting multidrug resistance. Conclusions. Smear and culture conversion in treated tuberculosis patients takes longer than is conventionally believed, even with fully susceptible disease, and must be accounted for in tuberculosis treatment and prevention programs. Persistent day 60 smear positivity is a poor predictor of multidrug resistance. The industrialized-world convention of universal baseline DST for tuberculosis patients should become the standard of care in multidrug resistance-affected resource-limited settings.

BACKGROUND: The microscopic observation drug susceptibility (MODS) assay for rapid, low-cost detection of tuberculosis and multidrug resistant tuberculosis depends upon visualization of the characteristic cording colonies of Mycobacterium tuberculosis in liquid media. This has conventionally required an inverted light microscope in order to inspect the MODS culture plates from below. Few tuberculosis laboratories have this item and the capital cost of $5,000 for a high-end microscope could be a significant obstacle to MODS roll-out. METHODOLOGY: We hypothesized that the precise definition provided by costly high-specification inverted light microscopes might not be necessary for pattern recognition. SIGNIFICANCE: In this work we describe the development of a low-cost artesenal inverted microscope that can operate in both a standard or digital mode to effectively replace the expensive commercial inverted light microscope, and an integrated system that could permit a local and remote diagnosis of tuberculosis.

The low-cost Microscopic Observation Drug Susceptibility (MODS) assay is a non-proprietary test that delivers rapid and accurate diagnosis of tuberculosis (TB) and multidrug-resistant TB. Although methodologically straightforward, implementation is challenging in isolated settings where personnel trained in plate reading are lacking. One affordable strategy to address this shortfall is the use of mobile phones, first to transmit images captured by an inverted microscope to a remote site where pattern recognition is performed by trained personnel, and second to receive the resulting output of this analysis. Such a system could be used for training of laboratory personnel through distance learning, resolution of equivocal appearances and quality assurance.

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections. Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains. The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB. The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB. In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.

BACKGROUND: New diagnostic tools are urgently needed to interrupt the transmission of tuberculosis and multidrug-resistant tuberculosis. Rapid, sensitive detection of tuberculosis and multidrug-resistant tuberculosis in sputum has been demonstrated in proof-of-principle studies of the microscopic-observation drug-susceptibility (MODS) assay, in which broth cultures are examined microscopically to detect characteristic growth. METHODS: In an operational setting in Peru, we investigated the performance of the MODS assay for culture and drug-susceptibility testing in three target groups: unselected patients with suspected tuberculosis, prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis, and unselected hospitalized patients infected with the human immunodeficiency virus. We compared the MODS assay head-to-head with two reference methods: automated mycobacterial culture and culture on Löwenstein-Jensen medium with the proportion method. RESULTS: Of 3760 sputum samples, 401 (10.7%) yielded cultures positive for Mycobacterium tuberculosis. Sensitivity of detection was 97.8% for MODS culture, 89.0% for automated mycobacterial culture, and 84.0% for Löwenstein-Jensen culture (P

An accurate urine test for pulmonary tuberculosis (TB), affecting 9.6 million patients worldwide, is critically needed for surveillance and treatment management. Past attempts failed to reliably detect the mycobacterial glycan antigen lipoarabinomannan (LAM), a marker of active TB, in HIV-negative, pulmonary TB–infected patients’ urine (85% of 9.6 million patients). We apply a copper complex dye within a hydrogel nanocage that captures LAM with very high affinity, displacing interfering urine proteins. The technology was applied to study pretreatment urine from 48 Peruvian patients, all negative for HIV, with microbiologically confirmed active pulmonary TB. LAM was quantitatively measured in the urine with a sensitivity of >95% and a specificity of >80% (n = 101) in a concentration range of 14 to 2000 picograms per milliliter, as compared to non-TB, healthy and diseased, age-matched controls (evaluated by receiver operating characteristic analysis; area under the curve, 0.95; 95% confidence interval, 0.9005 to 0.9957). Urinary LAM was elevated in patients with a higher mycobacterial burden (n = 42), a higher proportion of weight loss (n = 37), or cough (n = 50). The technology can be configured in a variety of formats to detect a panel of previously undetectable very-low-abundance TB urinary analytes. Eight of nine patients who were smear-negative and culture-positive for TB tested positive for urinary LAM. This technology has broad implications for pulmonary TB screening, transmission control, and treatment management for HIV-negative patients., Tuberculosis is no longer on the LAM Lipoarabinomannan (LAM) is a component of the cell wall shed by Mycobacterium tuberculosis, the bacteria responsible for tuberculosis, an infection mainly affecting the lungs. LAM can be detected in urine samples from patients coinfected with HIV, but current LAM detection methods have failed for HIV-negative patients. Using hydrogel “nanocage” nanoparticles and a chemical bait with high affinity for LAM, Paris et al. showed that patients negative for HIV with active tuberculosis infections had detectably higher concentrations of LAM in their urine than patients without active tuberculosis infections. Nanocages could also be used to detect cytokines and other antigens present in low concentrations in urine, demonstrating the versatility of the technology as a method to detect and monitor infections.