Search

ABSTRACT Xanthomonas citri subsp. citri (Xcc) is a bacterium that causes citrus canker, an economically important disease that results in premature fruit drop and reduced yield of fresh fruit. In this study, we demonstrated the involvement of XanB, an enzyme with phosphomannose isomerase (PMI) and guanosine diphosphate-mannose pyrophosphorylase (GMP) activities, in Xcc pathogenicity. Additionally, we found that XanB inhibitors protect the host against Xcc infection. Besides being deficient in motility, biofilm production, and ultraviolet resistance, the xanB deletion mutant was unable to cause disease, whereas xanB complementation restored wild-type phenotypes. XanB homology modeling allowed in silico virtual screening of inhibitors from databases, three of them being suitable in terms of absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties, which inhibited GMP (but not PMI) activity of the Xcc recombinant XanB protein in more than 50%. Inhibitors reduced citrus canker severity up to 95%, similarly to copper-based treatment. xanB is essential for Xcc pathogenicity, and XanB inhibitors can be used for the citrus canker control.IMPORTANCEXcc causes citrus canker, a threat to citrus production, which has been managed with copper, being required a more sustainable alternative for the disease control. XanB was previously found on the surface of Xcc, interacting with the host and displaying PMI and GMP activities. We demonstrated by xanB deletion and complementation that GMP activity plays a critical role in Xcc pathogenicity, particularly in biofilm formation. XanB homology modeling was performed, and in silico virtual screening led to carbohydrate-derived compounds able to inhibit XanB activity and reduce disease symptoms by 95%. XanB emerges as a promising target for drug design for control of citrus canker and other economically important diseases caused by Xanthomonas sp.

Abstract Background To date, real-world evidence on persistence to anti-calcitonin gene-related peptide (anti-CGRP) monoclonal antibodies (mAbs) or onabotulinumtoxinA have excluded eptinezumab. This retrospective cohort study was performed to compare treatment persistency among patients with migraine on anti-CGRP mAbs (erenumab, fremanezumab, galcanezumab, or eptinezumab) or onabotulinumtoxinA. Methods This retrospective study used IQVIA PharmMetrics data. Adult patients with migraine treated with an anti-CGRP mAb or onabotulinumtoxinA who had 12 months of continuous insurance enrollment before starting treatment were included. A “most recent treatment episode” analysis was used in which the most recent episode was defined as the latest treatment period with the same drug (anti-CGRP mAb or onabotulinumtoxinA) without a ≥ 15-day gap in medication supply on/after June 25, 2020, to December 31, 2021. Patients were indexed at the start of their most recent episode. Patients were considered non-persistent and discontinued the therapy associated with their most recent episode if there was ≥ 15-day gap in medication supply. A Cox proportional-hazards model estimated the discontinuation hazard between treatments. The gap periods and cohort definition were varied in sensitivity analyses. Results The study included 66,576 patients (median age 46 years, 88.6% female). More eptinezumab-treated patients had chronic migraine (727/1074), ≥ 3 previous acute (323/1074) or preventive (333/1074) therapies, and more prior treatment episodes (3) than other treatment groups. Based on a 15-day treatment gap, patients on subcutaneous anti-CGRP mAbs had a 32% (95% CI: 1.19, 1.49; erenumab), 42% (95% CI: 1.27, 1.61; galcanezumab), and 58% (95% CI: 1.42, 1.80; fremanezumab) higher discontinuation hazard than those receiving eptinezumab, with this relationship attenuated, but still statistically significant based on 30-day and 60-day treatment gaps. There was no significant difference in the discontinuation hazard between eptinezumab and onabotulinumtoxinA. Based on a 15-day treatment gap among patients who newly initiated therapy, the discontinuation hazard of subcutaneous anti-CGRP mAbs remained significantly higher compared to eptinezumab and onabotulinumtoxinA. Conclusion Patients treated with eptinezumab demonstrated persistency that was higher than subcutaneous anti-CGRP mAbs and similar to onabotulinumtoxinA. Graphical Abstract

BackgroundAccreditation of graduate academic programs in clinical research requires demonstration of program achievement of Joint Task Force for Clinical Trial Competence-based standards. Evaluation of graduate programs include enrollment, student grades, skills-based outcomes, and completion rates, in addition to other measures. Standardized measures of competence would be useful.MethodsWe used the Competency Index for Clinical Research Professionals (CICRP), in a separate-sample pretest-posttest study to measure self-confidence or self-efficacy in clinical research competency comparing cohorts of students entering and completing a master’s degree program in clinical research across three semesters (summer 2021 – spring 2022). CICRP is a 20-item Likert scale questionnaire (0 = Not at all confident; 10 = extremely confident).ResultsThe study sample of 110 students (54 in the entry course, 56 in the exit course) showed overall 80.9% entered the program with only a baccalaureate degree and 55.5% had no prior experience in managing clinical trial research. Cronbach alpha for the instrument showed a high level of content validity (range 0.93–0.98). Median CICRP item rating range at entry was [1, 6] and at exit [7, 10]. Mean CICRP total score (sum of 20 items) at entry was 72.7 (SD 41.9) vs. 167.0 (SD 21.1) at exit (p

In this study, we aimed to numerically investigate the 2D laminar flow over a cylindrical body and performed vortex-induced vibration analyses on a circular cylinder of unit radius placed in a channel, with the cylinder assumed to be fixed. The cases of a cylinder under forced oscillation and three different scenarios of a freely oscillating cylinder were analyzed. The fluid domain dynamics were governed by the incompressible Navier–Stokes equations; however, the structural field was described using nonlinear elastodynamic equations. Fluid and solid domains were discretized with the finite volume method (FVM) in space and time. Predictions of hydrodynamic forces, namely lift and drag terms, were determined for each scenario. An increase in the Reynolds number caused an exponential increment in the lift force. In the case of a stabilized flow, the collective decrease in stiffness and damping decreased the maximal drag and lift factors. Furthermore, it was noticed that the lift factor was minimally altered by variations in damping and stiffness in comparison with the change in the drag factor. From these observations, it appears that the lift factor probably correlates with the cylinder’s structure and fluid properties.

Background Tuberculosis is estimated to cause 1.5 million deaths annually and is most common during the reproductive years. Despite that fact, we found that tuberculosis screening, prevention or care recommendations for people around the time of pregnancy were absent from some national policy recommendations and varied in others. Objectives To address the apparent gaps and inconsistencies in policy, we aim to design a systematic review and meta-analysis of the original research evidence informing tuberculosis care around the time of pregnancy. Methods With assistance from librarians at the Biomedical library of the University of Gothenburg, Pubmed, CINAHL and Scopus databases will be searched. Search terms will aim to identify studies generating original research evidence informing care for tuberculosis around the time of pregnancy. Evidence may include: the outcome of TB and/or of pregnancy; the cost-effectiveness or acceptability of any intervention; the sensitivity and specificity of any assessment, selection, diagnostic or test criterion. The output from these literature searches will be screened by two independent reviewers to select the eligible studies for inclusion. Discrepancies will be resolved with a third reviewer. Firstly, publications that provide contextual data will be tabulated, summarising their main contributions. Secondly, studies that provide evidence directly guiding patient care will be our focus and will be considered to be key. The key studies will be subject to quality assessment, data extraction and when possible, meta-analysis. Conclusions This systematic review and meta-analysis aims to guide policy, practice and future research priorities concerning tuberculosis care around the time of pregnancy.

This study focuses on the impact of the sulfur vacancies on the photocatalytic response of the ZnS nanocrystals synthesized by solvothermal method varying the concentration of zinc acetate/thiourea precursors. XRD patterns show that these samples have a hexagonal structure with different degrees of crystallinity, varying the crystallite size from 2.48 to 2.85 nm. The UV-Vis data reveals an absorption peak (at about 320 nm) characteristic of ZnS nanocrystals. As a result, a decrease in the bandgap value of these materials was observed from 3.78 to 3.62 eV. In principle, a comparison of these results and theoretical calculations reveals the formation of intermediate levels inside the bandgap due to structural polarization. These findings also corroborate the zeta potential measured for these samples, evidenced by an increase of positive charge of ZnS surfaces. Also, the low Miller-index surfaces, such as (100), (110) and (0001), were investigated by periodic density functional theory calculations, in nice agreement with the experimental data. A photocatalysis mechanism was investigated and confirmed the formation of reactive oxygen species.

Mining began in the Picher field, in the Oklahoma and Kansas portion of the Tri-State Lead-Zinc Mining District in the United States, during the 1900s and ceased in the 1970s, producing an estimated 1.5 million metric tons (m-tons) of lead and 8 million m-tons of zinc. Over 400 historical maps of the underground mine workings were compiled into a single, easily editable map. This map was used to create 3D renderings for calculation of underground mine workings area and volume estimates. The workings have an estimated volume of 9,870 ha-meters (80,000 ac-ft), covering an area of 1,440 hectares (3,560 acres). The map and subsequent calculations should be considered to be based on the minimum extent of the mining field due to the likelihood that many historical maps were likely lost or destroyed. The format of the map allows for continuous updates as new information becomes available.

Background: Some sputum smear microscopy protocols recommend placing filter paper over sputum smears during staining for Mycobacterium tuberculosis (TB). We found no published evidence assessing whether this is beneficial. We aimed to evaluate the effect of filter paper on sputum smear microscopy results. Methods: Sputum samples were collected from 30 patients with confirmed pulmonary TB and 4 healthy control participants. From each sputum sample, six smears (204 smears in total) were prepared for staining with Ziehl-Neelsen (ZN), auramine or viability staining with fluorescein diacetate (FDA). Half of the slides subjected to each staining protocol were randomly selected to have Whatman grade 3 filter paper placed over the dried smears prior to stain application and removed prior to stain washing. The counts of acid-fast bacilli (AFB) and precipitates per 100 high-power microscopy fields of view, and the proportion of smear that appeared to have been washed away were recorded. Statistical analysis used a linear regression model adjusted by staining technique with a random effects term to correct for between-sample variability. Results: The inclusion of filter paper in the staining protocol significantly decreased microscopy positivity independent of staining with ZN, auramine or FDA (p=0.01). Consistent with this finding, there were lower smear grades in slides stained using filter paper versus without (p=0.04), and filter paper use reduced AFB counts by 0.28 logarithms (95% confidence intervals, CI=0.018, 0.54, p=0.04) independent of staining technique. In all analyses, auramine was consistently more sensitive with higher AFB counts versus ZN (p=0.001), whereas FDA had lower sensitivity and lower AFB counts (p

Background: COVID-19 variants threaten health globally. Despite improving vaccines and treatments, there is an urgent need for alternative strategies to prevent or reduce the severity of COVID-19. Potential strategies include probiotics, which are safe, inexpensive, globally available and have been studied previously in relation to respiratory infections. Methods: We performed a systematic review and meta-analyses of experimental, trial or observational research evidence evaluating probiotics compared with control groups for preventing or treating COVID-19. We searched PubMed, ProQuest, Google Scholar and Web of Science bibliographic databases for studies published until December 6, 2021. We then performed meta-analyses for outcomes reported consistently across studies. Outcomes reported inconsistently or not amenable to meta-analysis were compared descriptively. Results: We identified six eligible studies, which were all published in 2020 and 2021: one randomized controlled trial and five retrospective cohort studies. The only randomized controlled trial reported that groups that ingested probiotics compared with control groups that did not ingest probiotics did not differ significantly with respect to death, severe disease requiring admission to an intensive care unit or disease progression (all p>0.5). The five retrospective cohort studies reported various apparently beneficial and harmful COVID-19 outcome associations with probiotic ingestion. Meta-analyses revealed no significant associations between probiotic use and death, severe disease, or disease progression caused by COVID-19. Descriptive data revealed that probiotic ingestion was associated with a trend towards worsened duration of hospital stay, improvements in measures of respiratory condition and worsened disease duration. The evidence for these contradictory associations was weak because all studies were prone to bias and none were considered to be of high quality. Conclusions: Current evidence does not suggest that probiotics affect COVID-19 severity or mortality. However, additional higher quality studies need to be conducted to definitively determine if probiotics would be a useful adjunctive treatment for COVID-19.

Background: People with tuberculosis disease and their household members may suffer direct out-of-pocket expenses and indirect costs of lost income. These tuberculosis-related costs can worsen poverty, make tuberculosis treatment completion unaffordable, impair quality of life and increase the risk of death. Costs due to tuberculosis are usually defined as catastrophic if they exceed 20% of the pre-disease annual household income. The World Health Organisation strategy to “End TB” and the United Nations Sustainable Development Goals include the target that no households should face catastrophic costs due to tuberculosis. However, there is limited evidence and policy concerning how this global priority of eliminating catastrophic costs due to tuberculosis should be achieved. This systematic review and meta-analysis aims to address this knowledge gap. Methods: Publications assessing interventions that aimed to eliminate catastrophic costs will be identified by searching three electronic databases (PubMed, Scopus and Web of Science) together with reference lists from pertinent publications. We will screen eligible studies, extract data, and assess the risk of bias with the quality assessment tool from the National Heart, Lung, and Blood Institute. Discrepancies will be resolved by discussion between the reviewers. If we find sufficient comparable studies quantifying strategies to eliminate catastrophic costs then a meta-analysis will be performed. This systematic review and meta-analysis is registered with the PROSPERO database (CRD42022292410). Conclusion: This systematic review and meta-analysis aims to rigorously assess the evidence for strategies to eliminate catastrophic costs due to tuberculosis.

Objective: Developmental education provides postsecondary students requisite skills to take credit-bearing coursework. Yet, racial disparities exist in which students are most likely to be placed into developmental education. The present study investigates how multiple measures of students' mathematics background ameliorate racial disparities in developmental math outcomes among U.S. community college students. Methods: The High School Longitudinal Study of 2009 was used to identify sample participants (n = 3,650) who attended a 2-year postsecondary institution (community college) within 3 years of high school graduation. A series of logistic regression models were employed with developmental math outcomes as dependent variables predicted by multiple measures of student's math background and demographic covariates. Results: Compared to White students, Black and Hispanic students had poorer developmental math outcomes; however, inclusion of multiple measures of high school math experiences reduced racial disparities. Multiple measures significantly reduced developmental math enrollment by 13% for Black students and 15% for Hispanic students, improved developmental math passing rates for Black students by 25% and Hispanic students by 25%, and improved passing rates for the first college-credit bearing math course for Hispanic students by 73%. Conclusion: The present study highlights how multiple measures of students' math background might be an important lever for more racially-equitable placement processes. For Black and Hispanic students, the most influential high school measures were high school math GPA, highest high school math course, and math motivation. More work is needed to fine-tune how multiple measures can be used to increase access and equitable outcomes for students.

Motivational regulation, or the way learners purposefully initiate, maintain, or supplement their willingness for task engagement and completion, has been an important area of research in educational psychology. However, despite the surge of research on this topic, it remains unclear how specific motivational regulation strategies relate to learners' academic performance, motivation, and other forms of self-regulation. Based on findings from 55 studies (67 unique samples), we found a range of positive correlations among 10 types and subtypes of motivational regulation strategies and the following variables: academic achievement (rs = 0.01-0.15), motivation (effort, rs = 0.11-0.52; value perceptions, rs = 0.21-0.35; and competence beliefs, rs = 0.22-0.40), and self-regulation (cognitive, rs = 0.28-0.51; behavioral, rs = 0.10-0.33). Moderator results indicated that the magnitudes of associations among motivational regulation strategies and achievement and self-regulation varied by grade level and geographic region. Intercorrelations among motivational regulation strategies indicated wide-ranging degrees of overlap across strategies. Using meta-analytic structural equation modeling, we explored how motivational regulation strategies indirectly related to achievement through motivational beliefs and how self-regulatory factors were precursors to motivational regulation. Our meta-analytic findings shed light upon which motivational regulation strategies might be most adaptive for academic achievement, motivation, and self-regulation, and identified for whom and under what circumstances specific strategies can be most adaptively used.

Background: Appropriate technology tests are needed for Mycobacterium tuberculosis drug-susceptibility testing (DST) in resource-constrained settings. This study was performed to evaluate the MDR/XDR-TB Colour Test (a colour platethin-layer agar test; TB-CX) for M. tuberculosis DST by directly testing sputum at University of Gondar Hospital. Methods: Sputum samples were each divided into two aliquots. One aliquot was mixed with disinfectant and applied directly to the TB-CX quadrant petri-plate containing culture medium with and without isoniazid, rifampicin, or ciprofloxacin. Concurrently, the other aliquot was decontaminated with sodium hydroxide, centrifuged, and cultured on Lӧwenstein–Jensen medium; the stored M. tuberculosis isolates were then sub-cultured in BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 for reference DST. Results: The TB-CX test yielded DST results for 94% (123/131) of positive samples. For paired DST results, the median number of days from sputum processing to DST was 12 for TB-CX versus 35 for LJ-MGIT (p

AbstractIntroduction:There is a clear need to educate and train the clinical research workforce to conduct scientifically sound clinical research. Meeting this need requires the creation of tools to assess both an individual’s preparedness to function efficiently in the clinical research enterprise and tools to evaluate the quality and effectiveness of programs that are designed to educate and train clinical research professionals. Here we report the development and validation of a competency self-assessment entitled the Competency Index for Clinical Research Professionals, version II (CICRP-II).Methods:CICRP-II was developed using data collected from clinical research coordinators (CRCs) participating in the “Development, Implementation and Assessment of Novel Training In Domain-Based Competencies” (DIAMOND) project at four clinical and translational science award (CTSA) hubs and partnering institutions.Results:An exploratory factor analysis (EFA) identified a two-factor structure: the first factor measures self-reported competence to perform Routine clinical research functions (e.g., good clinical practice regulations (GCPs)), while the second factor measures competence to perform Advanced clinical functions (e.g., global regulatory affairs). We demonstrate the between groups validity by comparing CRCs working in different research settings.Discussion:The excellent psychometric properties of CICRP-II and its ability to distinguish between experienced CRCs at research-intensive CTSA hubs and CRCs working in less-intensive community-based sites coupled with the simplicity of alternative methods for scoring respondents make it a valuable tool for gauging an individual’s perceived preparedness to function in the role of CRC as well as an equally valuable tool to evaluate the value and effectiveness of clinical research education and training programs.

BackgroundWhole blood mycobacterial growth assays (WBMGA) quantify mycobacterial growth in fresh blood samples and may have potential for assessing tuberculosis vaccines and identifying individuals at risk of tuberculosis. We evaluated the evidence for the underlying assumption that in vitro WBMGA results can predict in vivo tuberculosis susceptibility.MethodsA systematic search was done for studies assessing associations between WBMGA results and tuberculosis susceptibility. Meta-analyses were performed for eligible studies by calculating population-weighted averages.ResultsNo studies directly assessed whether WBMGA results predicted tuberculosis susceptibility. 15 studies assessed associations between WBMGA results and proven correlates of tuberculosis susceptibility, which we divided in two categories. Firstly, WBMGA associations with factors believed to reduce tuberculosis susceptibility were statistically significant in all eight studies of: BCG vaccination; vitamin D supplementation; altitude; and HIV-negativity/therapy. Secondly, WBMGA associations with probable correlates of tuberculosis susceptibility were statistically significant in three studies of tuberculosis disease, in a parasitism study and in two of the five studies of latent tuberculosis infection. Meta-analyses for associations between WBMGA results and BCG vaccination, tuberculosis infection, tuberculosis disease and HIV infection revealed consistent effects. There was considerable methodological heterogeneity.ConclusionsThe study results generally showed significant associations between WBMGA results and correlates of tuberculosis susceptibility. However, no study directly assessed whether WBMGA results predicted actual susceptibility to tuberculosis infection or disease. We recommend optimization and standardization of WBMGA methodology and prospective studies to determine whether WBMGA predict susceptibility to tuberculosis disease.

ABSTRACT The filamentous fungus Aspergillus fumigatus can cause a distinct set of clinical disorders in humans. Invasive aspergillosis (IA) is the most common life-threatening fungal disease of immunocompromised humans. The mitogen-activated protein kinase (MAPK) signaling pathways are essential to the adaptation to the human host. Fungal cell survival is highly dependent on the organization, composition, and function of the cell wall. Here, an evaluation of the global A. fumigatus phosphoproteome under cell wall stress caused by the cell wall-damaging agent Congo red (CR) revealed 485 proteins potentially involved in the cell wall damage response. Comparative phosphoproteome analyses with the ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutant strains from the osmotic stress MAPK cascades identify their additional roles during the cell wall stress response. Our phosphoproteomics allowed the identification of novel kinases and transcription factors (TFs) involved in osmotic stress and in the cell wall integrity (CWI) pathway. Our global phosphoproteome network analysis showed an enrichment for protein kinases, RNA recognition motif domains, and the MAPK signaling pathway. In contrast to the wild-type strain, there is an overall decrease of differentially phosphorylated kinases and phosphatases in ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutants. We constructed phosphomutants for the phosphorylation sites of several proteins differentially phosphorylated in the wild-type and mutant strains. For all the phosphomutants, there is an increase in the sensitivity to cell wall-damaging agents and a reduction in the MpkA phosphorylation upon CR stress, suggesting these phosphosites could be important for the MpkA modulation and CWI pathway regulation. IMPORTANCE Aspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. The mitogen-activated protein kinase (MAPK) signaling pathways are essential for fungal adaptation to the human host. Fungal cell survival, fungicide tolerance, and virulence are highly dependent on the organization, composition, and function of the cell wall. Upon cell wall stress, MAPKs phosphorylate multiple target proteins involved in the remodeling of the cell wall. Here, we investigate the global phosphoproteome of the ΔsakA and ΔmpkC A. fumigatus and high-osmolarity glycerol (HOG) pathway MAPK mutants upon cell wall damage. This showed the involvement of the HOG pathway and identified novel protein kinases and transcription factors, which were confirmed by fungal genetics to be involved in promoting tolerance of cell wall damage. Our results provide understanding of how fungal signal transduction networks modulate the cell wall. This may also lead to the discovery of new fungicide drug targets to impact fungal cell wall function, fungicide tolerance, and virulence.

This paper reports findings from a research study at The Open University, UK into the quality of distance learners' online exam experience and the differences in experience between online (pandemic) and in-person (pre-pandemic) modes of examination. Our research responds to the ongoing need for greater insight into the exam experience and is?uniquely positioned in two ways. First, we made use of a robust reference dataset collected before the COVID-19 pandemic and compared this with a second survey administered a year after the pandemic started; second, we asked students about their experience preparing and revising for the exam as well as the exam itself. Exam revision represents an important transitional period for learners. Our results show that, overall, the shift to online remote exams did not impact the quality of distance learners' experience of revising for exams or taking the exam itself. We found no significant change in the revision experience across six of eight measures, including the learning benefits of learning while revising, enjoyment, and support. However, students reported feeling less anxious when revising for online exams. The quality of the exam experience itself was largely unaffected by the move from in-person to online remote exams. No significant differences were found for seven of the nine measures of exam experience. However, we found satisfaction with the exam environment was significantly higher for online exams and that learners felt the online exam was harder than they expected. Age and gender differences are also explored.

Utilizing a novel computerized task, we aimed to examine whether social anxiety symptoms would be related to individual differences in facial emotion recognition (FER) in a sample of autistic male adolescents and young adults without intellectual disability. Results indicated that social anxiety and IQ predicted poorer FER, irrespective of specific emotion type. When probing specific effects within emotion and condition types, social anxiety impacted surprise and disgust FER during a "truncated" viewing condition and not "full viewing" condition. Collectively, results suggest that social anxiety in autism may play a larger role in FER than previously thought. Future work should consider the role of social anxiety within autism as a factor that may meaningfully relate to FER assessment and intervention.

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.

BackgroundDrug susceptibility testing for Mycobacterium tuberculosis (MTB) is difficult to perform in resource-limited settings where Acid Fast Bacilli (AFB) smears are commonly used for disease diagnosis and monitoring. We developed a simple method for extraction of MTB DNA from AFB smears for sequencing-based detection of mutations associated with resistance to all first and several second-line anti-tuberculosis drugs.MethodsWe isolated MTB DNA by boiling smear content in a Chelex solution, followed by column purification. We sequenced PCR-amplified segments of the rpoB, katG, embB, gyrA, gyrB, rpsL, and rrs genes, the inhA, eis, and pncA promoters and the entire pncA gene.ResultsWe tested our assay on 1,208 clinically obtained AFB smears from Ghana (n = 379), Kenya (n = 517), Uganda (n = 262), and Zambia (n = 50). Coverage depth varied by target and slide smear grade, ranging from 300X to 12000X on average. Coverage of ≥20X was obtained for all targets in 870 (72%) slides overall. Mono-resistance (5.9%), multi-drug resistance (1.8%), and poly-resistance (2.4%) mutation profiles were detected in 10% of slides overall, and in over 32% of retreatment and follow-up cases.ConclusionThis rapid AFB smear DNA-based method for determining drug resistance may be useful for the diagnosis and surveillance of drug-resistant tuberculosis.