The number of privately treated tuberculosis cases in India: an estimation from drug sales data

Background Understanding the amount of tuberculosis managed by the private sector in India is crucial to understanding the true burden of the disease in the country, and thus globally. In the absence of quality surveillance data on privately treated patients, commercial drug sales data offer an empirical foundation for disease burden estimation. Methods We used a large, nationally representative commercial dataset on sales of 189 anti-tuberculosis products available in India to calculate the amount of anti-tuberculosis treatment in the private sector in 2013–14. We corrected estimates using validation studies that audited prescriptions against tuberculosis diagnosis, and estimated uncertainty using Monte Carlo simulation. To address implications for numbers of patients with tuberculosis, we explored varying assumptions for average duration of tuberculosis treatment and accuracy of private diagnosis. Findings There were 17·793 million patient-months (95% credible interval 16·709 million to 19·841 million) of anti-tuberculosis treatment in the private sector in 2014, twice as many as the public sector. If 40–60% of private-sector tuberculosis diagnoses are correct, and if private-sector tuberculosis treatment lasts on average 2–6 months, this implies that 1·19–5·34 million tuberculosis cases were treated in the private sector in 2014 alone. The midpoint of these ranges yields an estimate of 2·2 million cases, two to three times higher than currently assumed. Interpretation India's private sector is treating an enormous number of patients for tuberculosis, appreciably higher than has been previously recognised. Accordingly, there is a re-doubled need to address this burden and to strengthen surveillance. Tuberculosis burden estimates in India and worldwide require revision

Factors Associated with Tuberculosis and Rifampicin-Resistant Tuberculosis amongst Symptomatic Patients in India: A Retrospective Analysis

Background Tuberculosis remains a major public health challenge for India. Various studies have documented different levels of TB and multi-drug resistant (MDR) TB among diverse groups of the population. In view of renewed targets set under the End TB strategy by 2035, there is an urgent need for TB diagnosis to be strengthened. Drawing on data from a recent, multisite study, we address key questions for TB diagnosis amongst symptomatics presenting for care: are there subgroups of patients that are more likely than others, to be positive for TB? In turn, amongst these positive cases, are there factors—apart from treatment history—that may be predictive for multi-drug resistance? Methods We used data from a multi-centric prospective demonstration study, conducted from March 2012 to December 2013 in 18 sub-district level TB programme units (TUs) in India and covering a population of 8.8 million. In place of standard diagnostic tests, upfront Xpert MTB/RIF testing was offered to all presumptive TB symptomatics. Here, using data from this study, we used logistic regression to identify association between risk factors and TB and Rifampicin-Resistant TB among symptomatics enrolled in the study. Results We find that male gender; history of TB treatment; and adult age compared with either children or the elderly are risk factors associated with high TB detection amongst symptomatics, across the TUs. While treatment history is found be a significant risk factor for rifampicin-resistant TB, elderly (65+ yrs) people have significantly lower risk than other age groups. However, pediatric TB cases have no less risk of rifampicin resistance as compared with adults (OR 1.23 (95% C.I. 0.85–1.76)). Similarly, risk of rifampicin resistance among both the genders was the same. These patterns applied across the study sites involved. Notably in Mumbai, amongst those patients with microbiological confirmation of TB, female patients showed a higher risk of having MDR-TB than male patients. Conclusion Our results cast fresh light on the characteristics of symptomatics presenting for care who are most likely to be microbiologically positive for TB, and for rifampicin resistance. The challenges posed by TB control are complex and multifactorial: evidence from diverse sources, including retrospective studies such as that addressed here, can be invaluable in informing future strategies to accelerate declines in TB burden

Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di- GMP) is a dynamic intracellular signaling molecule that plays a central role in the biofilm life cycle. Current methodologies for the quantification of c-di-GMP are typically based on chemical extraction, representing end point measurements. Chemical methodologies also fail to take into consideration the physiological heterogeneity of the biofilm and thus represent an average c-di-GMP concentration across the entire biofilm. To address these problems, a ratiometric, image-based quantification method has been developed based on expression of the green fluorescence protein (GFP) under the control of the c-di-GMPresponsive cdrA promoter (Rybtke, M. T., Borlee, B. R., Murakami, K., Irie, Y., Hentzer, M., Nielsen, T. E., Givskov, M., Parsek, M. R., and Tolker-Nielsen, T. (2012) Appl. Environ. Microbiol. 78, 5060-5069). The methodology uses the cyan fluorescent protein (CFP) as a biomass indicator and the GFP as a c-di-GMP reporter. Thus, the CFP/GFP ratio gives the effective c-di-GMP per biomass. A binary mask was applied to alleviate background fluorescence, and fluorescence was calibrated against known c-di-GMP concentrations. Using flow cells for biofilm formation, c-di-GMP showed a non-uniform distribution across the biofilm, with concentrated hot spots of c-di- GMP. Additionally, c-di-GMP was found to be localized at the outer boundary of mature colonies in contrast to a uniform distribution in early stage, small colonies. These data demonstrate the application of a method for the in situ, real time quantification of c-di-GMP and show that the amount of this biofilmregulating second messenger was dynamic with time and colony size, reflecting the extent of biofilm heterogeneity in real time

Bottom-up or top-down: unit cost estimation of tuberculosis diagnostic tests in India.

SETTING: Of 18 sites that participated in an implementation study of the Xpert® MTB/RIF assay in India, we selected five microscopy centres and two reference laboratories. OBJECTIVE: To obtain unit costs of diagnostic tests for tuberculosis (TB) and drug-resistant TB. DESIGN: Laboratories were purposely selected to capture regional variations and different laboratory types. Both bottom-up and the top-down methods were used to estimate unit costs. RESULTS: At the microscopy centres, mean bottom-up unit costs were respectively US$0.83 (range US$0.60-US$1.10) and US$12.29 (US$11.61-US$12.89) for sputum smear microscopy and Xpert. At the reference laboratories, mean unit costs were US$1.69 for the decontamination procedure, US$9.83 for a solid culture, US$11.06 for a liquid culture, US$29.88 for a drug susceptibility test, and US$18.18 for a line-probe assay. Top-down mean unit cost estimates were higher for all tests, and for sputum smear microscopy and Xpert these increased to respectively US$1.51 and US$13.58. The difference between bottom-up and top-down estimates was greatest for tests performed at the reference laboratories. CONCLUSION: These unit costs for TB diagnostics can be used to estimate resource requirements and cost-effectiveness in India, taking into account geographical location, laboratory type and capacity utilisation

Carbon starvation of Pseudomonas aeruginosa biofilms selects for dispersal insensitive mutants.

BACKGROUND: Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation. RESULTS: In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain. CONCLUSIONS: This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation

Risk factors for hospital admission with RSV bronchiolitis in England: a population-based birth cohort study.

OBJECTIVE: To examine the timing and duration of RSV bronchiolitis hospital admission among term and preterm infants in England and to identify risk factors for bronchiolitis admission. DESIGN: A population-based birth cohort with follow-up to age 1 year, using the Hospital Episode Statistics database. SETTING: 71 hospitals across England. PARTICIPANTS: We identified 296618 individual birth records from 2007/08 and linked to subsequent hospital admission records during the first year of life. RESULTS: In our cohort there were 7189 hospital admissions with a diagnosis of bronchiolitis, 24.2 admissions per 1000 infants under 1 year (95%CI 23.7-24.8), of which 15% (1050/7189) were born preterm (47.3 bronchiolitis admissions per 1000 preterm infants (95% CI 44.4-50.2)). The peak age group for bronchiolitis admissions was infants aged 1 month and the median was age 120 days (IQR = 61-209 days). The median length of stay was 1 day (IQR = 0-3). The relative risk (RR) of a bronchiolitis admission was higher among infants with known risk factors for severe RSV infection, including those born preterm (RR = 1.9, 95% CI 1.8-2.0) compared with infants born at term. Other conditions also significantly increased risk of bronchiolitis admission, including Down's syndrome (RR = 2.5, 95% CI 1.7-3.7) and cerebral palsy (RR = 2.4, 95% CI 1.5-4.0). CONCLUSIONS: Most (85%) of the infants who are admitted to hospital with bronchiolitis in England are born at term, with no known predisposing risk factors for severe RSV infection, although risk of admission is higher in known risk groups. The early age of bronchiolitis admissions has important implications for the potential impact and timing of future active and passive immunisations. More research is needed to explain why babies born with Down's syndrome and cerebral palsy are also at higher risk of hospital admission with RSV bronchiolitis

A review of RCTs in four medical journals to assess the use of imputation to overcome missing data in quality of life outcomes

Background: Randomised controlled trials (RCTs) are perceived as the gold-standard method for evaluating healthcare interventions, and increasingly include quality of life (QoL) measures. The observed results are susceptible to bias if a substantial proportion of outcome data are missing. The review aimed to determine whether imputation was used to deal with missing QoL outcomes. Methods: A random selection of 285 RCTs published during 2005/6 in the British Medical Journal, Lancet, New England Journal of Medicine and Journal of American Medical Association were identified. Results: QoL outcomes were reported in 61 (21%) trials. Six (10%) reported having no missing data, 20 (33%) reported ≤ 10% missing, eleven (18%) 11%–20% missing, and eleven (18%) reported >20% missing. Missingness was unclear in 13 (21%). Missing data were imputed in 19 (31%) of the 61 trials. Imputation was part of the primary analysis in 13 trials, but a sensitivity analysis in six. Last value carried forward was used in 12 trials and multiple imputation in two. Following imputation, the most common analysis method was analysis of covariance (10 trials). Conclusion: The majority of studies did not impute missing data and carried out a complete-case analysis. For those studies that did impute missing data, researchers tended to prefer simpler methods of imputation, despite more sophisticated methods being available.The Health Services Research Unit is funded by the Chief Scientist Office of the Scottish Government Health Directorate. Shona Fielding is also currently funded by the Chief Scientist Office on a Research Training Fellowship (CZF/1/31)

A review of RCTs in four medical journals to assess the use of imputation to overcome missing data in quality of life outcomes

Peer reviewedPublisher PD

Dendritic cells loaded with killed breast cancer cells induce differentiation of tumor-specific cytotoxic T lymphocytes

BACKGROUND: Early clinical trials, mostly in the setting of melanoma, have shown that dendritic cells (DCs) expressing tumor antigens induce some immune responses and some clinical responses. A major difficulty is the extension to other tumors, such as breast carcinoma, for which few defined tumor-associated antigens are available. We have demonstrated, using both prostate carcinoma and melanoma as model systems, that DCs loaded with killed allogeneic tumor cell lines can induce CD8(+ )T cells to differentiate into cytotoxic T lymphocytes (CTLs) specific for shared tumor antigens. METHODS: The present study was designed to determine whether DCs would capture killed breast cancer cells and present their antigens to autologous CD4(+ )and CD8(+ )T cells. RESULTS: We show that killed breast cancer cells are captured by immature DCs that, after induced maturation, can efficiently present MHC class I and class II peptides to CD8(+ )and CD4(+ )T lymphocytes. The elicited CTLs are able to kill the target cells without a need for pretreatment with interferon gamma. CTLs can be obtained by culturing the DCs loaded with killed breast cancer cells with unseparated peripheral blood lymphocytes, indicating that the DCs can overcome any potential inhibitory effects of breast cancer cells. CONCLUSION: Loading DCs with killed breast cancer cells may be considered a novel approach to breast cancer immunotherapy and to identification of shared breast cancer antigens