Reduction of malaria prevalence by indoor residual spraying: a meta-regression analysis.

Indoor residual spraying (IRS) has become an increasingly popular method of insecticide use for malaria control, and many recent studies have reported on its effectiveness in reducing malaria burden in a single community or region. There is a need for systematic review and integration of the published literature on IRS and the contextual determining factors of its success in controlling malaria. This study reports the findings of a meta-regression analysis based on 13 published studies, which were chosen from more than 400 articles through a systematic search and selection process. The summary relative risk for reducing malaria prevalence was 0.38 (95% confidence interval = 0.31-0.46), which indicated a risk reduction of 62%. However, an excessive degree of heterogeneity was found between the studies. The meta-regression analysis indicates that IRS is more effective with high initial prevalence, multiple rounds of spraying, use of DDT, and in regions with a combination of Plasmodium falciparum and P. vivax malaria.

Factors associated with mortality in transplant patients with invasive aspergillosis.

BACKGROUND: Invasive aspergillosis (IA) is an important cause of morbidity and mortality in hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) recipients. The purpose of this study was to evaluate factors associated with mortality in transplant patients with IA. METHODS: Transplant patients from 23 US centers were enrolled from March 2001 to October 2005 as part of the Transplant Associated Infection Surveillance Network. IA cases were identified prospectively in this cohort through March 2006, and data were collected. Factors associated with 12-week all-cause mortality were determined by logistic regression analysis and Cox proportional hazards regression. RESULTS: Six-hundred forty-two cases of proven or probable IA were evaluated, of which 317 (49.4%) died by the study endpoint. All-cause mortality was greater in HSCT patients (239 [57.5%] of 415) than in SOT patients (78 [34.4%] of 227; P<.001). Independent poor prognostic factors in HSCT patients were neutropenia, renal insufficiency, hepatic insufficiency, early-onset IA, proven IA, and methylprednisolone use. In contrast, white race was associated with decreased risk of death. Among SOT patients, hepatic insufficiency, malnutrition, and central nervous system disease were poor prognostic indicators, whereas prednisone use was associated with decreased risk of death. Among HSCT or SOT patients who received antifungal therapy, use of an amphotericin B preparation as part of initial therapy was associated with increased risk of death. CONCLUSIONS: There are multiple variables associated with survival in transplant patients with IA. Understanding these prognostic factors may assist in the development of treatment algorithms and clinical trials.

Polymorphisms at the microRNA binding-site of the stem cell marker gene CD133 modify susceptibility to and survival of gastric cancer.

CD133 is one of the most common stem cell markers, and functional single nucleotide polymorphisms (SNPs) of CD133 may modulate its gene functions and thus cancer risk and patient survival. We hypothesized that potentially functional CD133 SNPs are associated with gastric cancer (GC) risk and survival. To test this hypothesis, we conducted a case-control study of 371 GC patients and 313 cancer-free controls frequency-matched by age, sex, and ethnicity. We genotyped four selected, potentially functional CD133 SNPs (rs2240688A>C, rs7686732C>G, rs10022537T>A, and rs3130C>T) and used logistic regression analysis for associations of these SNPs with GC risk and Cox hazards regression analysis for survival. We found that compared with the miRNA binding site rs2240688 AA genotype, AC + CC genotypes were associated with significantly increased GC risk (adjusted OR = 1.52, 95% CI = 1.09-2.13); for another miRNA binding site rs3130C>T SNP, the TT genotype was associated with significantly reduced GC risk (adjusted OR = 0.68, 95% CI = 0.48-0.97), compared with CC + CT genotypes. In all patients, the risk rs3130 TT variant genotype was significantly associated with overall survival (OS) (adjusted P(trend) = 0.016 and 0.007 under additive and recessive models, respectively). These findings suggest that these two CD133 miRNA binding site variants, rs2240688 and rs3130, may be potential biomarkers for genetic susceptibility to GC and possible predictors for survival in GC patients but require further validation by larger studies.

Polymorphisms in the kinesin-like factor 1 B gene and risk of epithelial ovarian cancer in Eastern Chinese women.

The kinesin-like factor 1 B (KIF1B) gene plays an important role in the process of apoptosis and the transformation and progression of malignant cells. Genetic variations in KIF1B may contribute to risk of epithelial ovarian cancer (EOC). In this study of 1,324 EOC patients and 1,386 cancer-free female controls, we investigated associations between two potentially functional single nucleotide polymorphisms in KIF1B and EOC risk by the conditional logistic regression analysis. General linear regression model was used to evaluate the correlation between the number of variant alleles and KIF1B mRNA expression levels. We found that the rs17401966 variant AG/GG genotypes were significantly associated with a decreased risk of EOC (adjusted odds ratio (OR) = 0.81, 95 % confidence interval (CI) = 0.68-0.97), compared with the AA genotype, but no associations were observed for rs1002076. Women who carried both rs17401966 AG/GG and rs1002076 AG/AA genotypes of KIF1B had a 0.82-fold decreased risk (adjusted 95 % CI = 0.69-0.97), compared with others. Additionally, there was no evidence of possible interactions between about-mentioned co-variants. Further genotype-phenotype correlation analysis indicated that the number of rs17401966 variant G allele was significantly associated with KIF1B mRNA expression levels (P for GLM = 0.003 and 0.001 in all and Chinese subjects, respectively), with GG carriers having the lowest level of KIF1B mRNA expression. Taken together, the rs17401966 polymorphism likely regulates KIF1B mRNA expression and thus may be associated with EOC risk in Eastern Chinese women. Larger, independent studies are warranted to validate our findings.

Referring heroin users from compulsory detoxification centers to community methadone maintenance treatment: a comparison of three models.

BACKGROUND: Both compulsory detoxification treatment and community-based methadone maintenance treatment (MMT) exist for heroin addicts in China. We aim to examine the effectiveness of three intervention models for referring heroin addicts released from compulsory detoxification centers to community methadone maintenance treatment (MMT) clinics in Dehong prefecture, Yunnan province, China. METHODS: Using a quasi-experimental study design, three different referral models were assigned to four detoxification centers. Heroin addicts were enrolled based on their fulfillment to eligibility criteria and provision of informed consent. Two months prior to their release, information on demographic characteristics, history of heroin use, and prior participation in intervention programs was collected via a survey, and blood samples were obtained for HIV testing. All subjects were followed for six months after release from detoxification centers. Multi-level logistic regression analysis was used to examine factors predicting successful referrals to MMT clinics. RESULTS: Of the 226 participants who were released and followed, 9.7% were successfully referred to MMT(16.2% of HIV-positive participants and 7.0% of HIV-negative participants). A higher proportion of successful referrals was observed among participants who received both referral cards and MMT treatment while still in detoxification centers (25.8%) as compared to those who received both referral cards and police-assisted MMT enrollment (5.4%) and those who received referral cards only (0%). Furthermore, those who received referral cards and MMT treatment while still in detoxification had increased odds of successful referral to an MMT clinic (adjusted OR = 1.2, CI = 1.1-1.3). Having participated in an MMT program prior to detention (OR = 1.5, CI = 1.3-1.6) was the only baseline covariate associated with increased odds of successful referral. CONCLUSION: Findings suggest that providing MMT within detoxification centers promotes successful referral of heroin addicts to community-based MMT upon their release.

Structured Illumination Microscopy and a Quantitative Image Analysis for the Detection of Positive Margins in a Pre-Clinical Genetically Engineered Mouse Model of Sarcoma.

Intraoperative assessment of surgical margins is critical to ensuring residual tumor does not remain in a patient. Previously, we developed a fluorescence structured illumination microscope (SIM) system with a single-shot field of view (FOV) of 2.1 × 1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 μm). The goal of this study was to test the utility of this technology for the detection of residual disease in a genetically engineered mouse model of sarcoma. Primary soft tissue sarcomas were generated in the hindlimb and after the tumor was surgically removed, the relevant margin was stained with acridine orange (AO), a vital stain that brightly stains cell nuclei and fibrous tissues. The tissues were imaged with the SIM system with the primary goal of visualizing fluorescent features from tumor nuclei. Given the heterogeneity of the background tissue (presence of adipose tissue and muscle), an algorithm known as maximally stable extremal regions (MSER) was optimized and applied to the images to specifically segment nuclear features. A logistic regression model was used to classify a tissue site as positive or negative by calculating area fraction and shape of the segmented features that were present and the resulting receiver operator curve (ROC) was generated by varying the probability threshold. Based on the ROC curves, the model was able to classify tumor and normal tissue with 77% sensitivity and 81% specificity (Youden's index). For an unbiased measure of the model performance, it was applied to a separate validation dataset that resulted in 73% sensitivity and 80% specificity. When this approach was applied to representative whole margins, for a tumor probability threshold of 50%, only 1.2% of all regions from the negative margin exceeded this threshold, while over 14.8% of all regions from the positive margin exceeded this threshold.

Autonomic cardiovascular dysregulation as a potential mechanism underlying depression and coronary artery bypass grafting surgery outcomes.

BACKGROUND: Coronary artery bypass grafting (CABG) is often used to treat patients with significant coronary heart disease (CHD). To date, multiple longitudinal and cross-sectional studies have examined the association between depression and CABG outcomes. Although this relationship is well established, the mechanism underlying this relationship remains unclear. The purpose of this study was twofold. First, we compared three markers of autonomic nervous system (ANS) function in four groups of patients: 1) Patients with coronary heart disease and depression (CHD/Dep), 2) Patients without CHD but with depression (NonCHD/Dep), 3) Patients with CHD but without depression (CHD/NonDep), and 4) Patients without CHD and depression (NonCHD/NonDep). Second, we investigated the impact of depression and autonomic nervous system activity on CABG outcomes. METHODS: Patients were screened to determine whether they met some of the study's inclusion or exclusion criteria. ANS function (i.e., heart rate, heart rate variability, and plasma norepinephrine levels) were measured. Chi-square and one-way analysis of variance were performed to evaluate group differences across demographic, medical variables, and indicators of ANS function. Logistic regression and multiple regression analyses were used to assess impact of depression and autonomic nervous system activity on CABG outcomes. RESULTS: The results of the study provide some support to suggest that depressed patients with CHD have greater ANS dysregulation compared to those with only CHD or depression. Furthermore, independent predictors of in-hospital length of stay and non-routine discharge included having a diagnosis of depression and CHD, elevated heart rate, and low heart rate variability. CONCLUSIONS: The current study presents evidence to support the hypothesis that ANS dysregulation might be one of the underlying mechanisms that links depression to cardiovascular CABG surgery outcomes. Thus, future studies should focus on developing and testing interventions that targets modifying ANS dysregulation, which may lead to improved patient outcomes.

Gender differences in the risk of HIV infection among persons reporting abstinence, monogamy, and multiple sexual partners in northern Tanzania.

BACKGROUND: Monogamy, together with abstinence, partner reduction, and condom use, is widely advocated as a key behavioral strategy to prevent HIV infection in sub-Saharan Africa. We examined the association between the number of sexual partners and the risk of HIV seropositivity among men and women presenting for HIV voluntary counseling and testing (VCT) in northern Tanzania. METHODOLOGY/ PRINCIPAL FINDINGS: Clients presenting for HIV VCT at a community-based AIDS service organization in Moshi, Tanzania were surveyed between November 2003 and December 2007. Data on sociodemographic characteristics, reasons for testing, sexual behaviors, and symptoms were collected. Men and women were categorized by number of lifetime sexual partners, and rates of seropositivity were reported by category. Factors associated with HIV seropositivity among monogamous males and females were identified by a multivariate logistic regression model. Of 6,549 clients, 3,607 (55%) were female, and the median age was 30 years (IQR 24-40). 939 (25%) females and 293 (10%) males (p<0.0001) were HIV seropositive. Among 1,244 (34%) monogamous females and 423 (14%) monogamous males, the risk of HIV infection was 19% and 4%, respectively (p<0.0001). The risk increased monotonically with additional partners up to 45% (p<0.001) and 15% (p<0.001) for women and men, respectively with 5 or more partners. In multivariate analysis, HIV seropositivity among monogamous women was most strongly associated with age (p<0.0001), lower education (p<0.004), and reporting a partner with other partners (p = 0.015). Only age was a significant risk factor for monogamous men (p = 0.0004). INTERPRETATION: Among women presenting for VCT, the number of partners is strongly associated with rates of seropositivity; however, even women reporting lifetime monogamy have a high risk for HIV infection. Partner reduction should be coupled with efforts to place tools in the hands of sexually active women to reduce their risk of contracting HIV.

Spatial distribution of bednet coverage under routine distribution through the public health sector in a rural district in Kenya.

Insecticide-treated nets (ITNs) are one of the most important and cost-effective tools for malaria control. Maximizing individual and community benefit from ITNs requires high population-based coverage. Several mechanisms are used to distribute ITNs, including health facility-based targeted distribution to high-risk groups; community-based mass distribution; social marketing with or without private sector subsidies; and integrating ITN delivery with other public health interventions. The objective of this analysis is to describe bednet coverage in a district in western Kenya where the primary mechanism for distribution is to pregnant women and infants who attend antenatal and immunization clinics. We use data from a population-based census to examine the extent of, and factors correlated with, ownership of bednets. We use both multivariable logistic regression and spatial techniques to explore the relationship between household bednet ownership and sociodemographic and geographic variables. We show that only 21% of households own any bednets, far lower than the national average, and that ownership is not significantly higher amongst pregnant women attending antenatal clinic. We also show that coverage is spatially heterogeneous with less than 2% of the population residing in zones with adequate coverage to experience indirect effects of ITN protection.

Correlates of poor health among orphans and abandoned children in less wealthy countries: the importance of caregiver health.

BACKGROUND: More than 153 million children worldwide have been orphaned by the loss of one or both parents, and millions more have been abandoned. We investigated relationships between the health of orphaned and abandoned children (OAC) and child, caregiver, and household characteristics among randomly selected OAC in five countries. METHODOLOGY: Using a two-stage random sampling strategy in 6 study areas in Cambodia, Ethiopia, India, Kenya, and Tanzania, the Positive Outcomes for Orphans (POFO) study identified 1,480 community-living OAC ages 6 to 12. Detailed interviews were conducted with 1,305 primary caregivers at baseline and after 6 and 12 months. Multivariable logistic regression models describe associations between the characteristics of children, caregivers, and households and child health outcomes: fair or poor child health; fever, cough, or diarrhea within the past two weeks; illness in the past 6 months; and fair or poor health on at least two assessments. PRINCIPAL FINDINGS: Across the six study areas, 23% of OAC were reported to be in fair or poor health; 19%, 18%, and 2% had fever, cough, or diarrhea, respectively, within the past two weeks; 55% had illnesses within the past 6 months; and 23% were in fair or poor health on at least two assessments. Female gender, suspected HIV infection, experiences of potentially traumatic events, including the loss of both parents, urban residence, eating fewer than 3 meals per day, and low caregiver involvement were associated with poorer child health outcomes. Particularly strong associations were observed between child health measures and the health of their primary caregivers. CONCLUSIONS: Poor caregiver health is a strong signal for poor health of OAC. Strategies to support OAC should target the caregiver-child dyad. Steps to ensure food security, foster gender equality, and prevent and treat traumatic events are needed.

Genetic variants in IGF-I, IGF-II, IGFBP-3, and adiponectin genes and colon cancer risk in African Americans and Whites.

PURPOSE: Evaluating genetic susceptibility may clarify effects of known environmental factors and also identify individuals at high risk. We evaluated the association of four insulin-related pathway gene polymorphisms in insulin-like growth factor-1 (IGF-I) (CA)( n ) repeat, insulin-like growth factor-2 (IGF-II) (rs680), insulin-like growth factor-binding protein-3 (IGFBP-3) (rs2854744), and adiponectin (APM1 rs1501299) with colon cancer risk, as well as relationships with circulating IGF-I, IGF-II, IGFBP-3, and C-peptide in a population-based study. METHODS: Participants were African Americans (231 cases and 306 controls) and Whites (297 cases, 530 controls). Consenting subjects provided blood specimens and lifestyle/diet information. Genotyping for all genes except IGF-I was performed by the 5'-exonuclease (Taqman) assay. The IGF-I (CA)(n) repeat was assayed by PCR and fragment analysis. Circulating proteins were measured by enzyme immunoassays. Odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated by logistic regression. RESULTS: The IGF-I (CA)( 19 ) repeat was higher in White controls (50 %) than African American controls (31 %). Whites homozygous for the IGF-I (CA)(19) repeat had a nearly twofold increase in risk of colon cancer (OR = 1.77; 95 % CI = 1.15-2.73), but not African Americans (OR = 0.73, 95 % CI 0.50-1.51). We observed an inverse association between the IGF-II Apa1 A-variant and colon cancer risk (OR = 0.49, 95 % CI 0.28-0.88) in Whites only. Carrying the IGFBP-3 variant alleles was associated with lower IGFBP-3 protein levels, a difference most pronounced in Whites (p-trend <0.05). CONCLUSIONS: These results support an association between insulin pathway-related genes and elevated colon cancer risk in Whites but not in African Americans.

Lognormal and gamma mixed negative binomial regression

In regression analysis of counts, a lack of simple and efficient algorithms for posterior computation has made Bayesian approaches appear unattractive and thus underdeveloped. We propose a lognormal and gamma mixed negative binomial (NB) regression model for counts, and present efficient closed-form Bayesian inference; unlike conventional Poisson models, the proposed approach has two free parameters to include two different kinds of random effects, and allows the incorporation of prior information, such as sparsity in the regression coefficients. By placing a gamma distribution prior on the NB dispersion parameter r, and connecting a log-normal distribution prior with the logit of the NB probability parameter p, efficient Gibbs sampling and variational Bayes inference are both developed. The closed-form updates are obtained by exploiting conditional conjugacy via both a compound Poisson representation and a Polya-Gamma distribution based data augmentation approach. The proposed Bayesian inference can be implemented routinely, while being easily generalizable to more complex settings involving multivariate dependence structures. The algorithms are illustrated using real examples. Copyright 2012 by the author(s)/owner(s).

Preoperative pain level and patient expectation predict hospital length of stay after total hip arthroplasty.

The purpose of this study was to identify preoperative predictors of length of stay after primary total hip arthroplasty in a patient population reflecting current trends toward shorter hospitalization and using readily obtainable factors that do not require scoring systems. A retrospective review of 112 consecutive patients was performed. High preoperative pain level and patient expectation of discharge to extended care facilities (ECFs) were the only significant multivariable predictors of hospitalization extending beyond 2 days (P=0.001 and P<0.001 respectively). Patient expectation remained significant after adjusting for Medicare's 3-day requirement for discharge to ECFs (P<0.001). The study was adequately powered to analyze the variables in the multivariable logistic regression model, which had a concordance index of 0.857.

Patient-provider communication, self-reported medication adherence, and race in a postmyocardial infarction population.

OBJECTIVES: Our objectives were to: 1) describe patient-reported communication with their provider and explore differences in perceptions of racially diverse adherent versus nonadherent patients; and 2) examine whether the association between unanswered questions and patient-reported medication nonadherence varied as a function of patients' race. METHODS: We conducted a cross-sectional analysis of baseline in-person survey data from a trial designed to improve postmyocardial infarction management of cardiovascular disease risk factors. RESULTS: Overall, 298 patients (74%) reported never leaving their doctor's office with unanswered questions. Among those who were adherent and nonadherent with their medications, 183 (79%) and 115 (67%) patients, respectively, never left their doctor's office with unanswered questions. In multivariable logistic regression, although the simple effects of the interaction term were different for patients of nonminority race (odds ratio [OR]: 2.16; 95% confidence interval [CI]: 1.19-3.92) and those of minority race (OR: 1.19; 95% CI: 0.54-2.66), the overall interaction effect was not statistically significant (P=0.24). CONCLUSION: The quality of patient-provider communication is critical for cardiovascular disease medication adherence. In this study, however, having unanswered questions did not impact medication adherence differently as a function of patients' race. Nevertheless, there were racial differences in medication adherence that may need to be addressed to ensure optimal adherence and health outcomes. Effort should be made to provide training opportunities for both patients and their providers to ensure strong communication skills and to address potential differences in medication adherence in patients of diverse backgrounds.

Micro-Anatomical Quantitative Imaging Towards Enabling Automated Diagnosis of Thick Tissues at the Point of Care

Histopathology is the clinical standard for tissue diagnosis. However, histopathology has several limitations including that it requires tissue processing, which can take 30 minutes or more, and requires a highly trained pathologist to diagnose the tissue. Additionally, the diagnosis is qualitative, and the lack of quantitation leads to possible observer-specific diagnosis. Taken together, it is difficult to diagnose tissue at the point of care using histopathology.

Several clinical situations could benefit from more rapid and automated histological processing, which could reduce the time and the number of steps required between obtaining a fresh tissue specimen and rendering a diagnosis. For example, there is need for rapid detection of residual cancer on the surface of tumor resection specimens during excisional surgeries, which is known as intraoperative tumor margin assessment. Additionally, rapid assessment of biopsy specimens at the point-of-care could enable clinicians to confirm that a suspicious lesion is successfully sampled, thus preventing an unnecessary repeat biopsy procedure. Rapid and low cost histological processing could also be potentially useful in settings lacking the human resources and equipment necessary to perform standard histologic assessment. Lastly, automated interpretation of tissue samples could potentially reduce inter-observer error, particularly in the diagnosis of borderline lesions.

To address these needs, high quality microscopic images of the tissue must be obtained in rapid timeframes, in order for a pathologic assessment to be useful for guiding the intervention. Optical microscopy is a powerful technique to obtain high-resolution images of tissue morphology in real-time at the point of care, without the need for tissue processing. In particular, a number of groups have combined fluorescence microscopy with vital fluorescent stains to visualize micro-anatomical features of thick (i.e. unsectioned or unprocessed) tissue. However, robust methods for segmentation and quantitative analysis of heterogeneous images are essential to enable automated diagnosis. Thus, the goal of this work was to obtain high resolution imaging of tissue morphology through employing fluorescence microscopy and vital fluorescent stains and to develop a quantitative strategy to segment and quantify tissue features in heterogeneous images, such as nuclei and the surrounding stroma, which will enable automated diagnosis of thick tissues.

To achieve these goals, three specific aims were proposed. The first aim was to develop an image processing method that can differentiate nuclei from background tissue heterogeneity and enable automated diagnosis of thick tissue at the point of care. A computational technique called sparse component analysis (SCA) was adapted to isolate features of interest, such as nuclei, from the background. SCA has been used previously in the image processing community for image compression, enhancement, and restoration, but has never been applied to separate distinct tissue types in a heterogeneous image. In combination with a high resolution fluorescence microendoscope (HRME) and a contrast agent acriflavine, the utility of this technique was demonstrated through imaging preclinical sarcoma tumor margins. Acriflavine localizes to the nuclei of cells where it reversibly associates with RNA and DNA. Additionally, acriflavine shows some affinity for collagen and muscle. SCA was adapted to isolate acriflavine positive features or APFs (which correspond to RNA and DNA) from background tissue heterogeneity. The circle transform (CT) was applied to the SCA output to quantify the size and density of overlapping APFs. The sensitivity of the SCA+CT approach to variations in APF size, density and background heterogeneity was demonstrated through simulations. Specifically, SCA+CT achieved the lowest errors for higher contrast ratios and larger APF sizes. When applied to tissue images of excised sarcoma margins, SCA+CT correctly isolated APFs and showed consistently increased density in tumor and tumor + muscle images compared to images containing muscle. Next, variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82% and 75%. The utility of this approach was further tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78% and 82%. The results indicate that SCA+CT can accurately delineate APFs in heterogeneous tissue, which is essential to enable automated and rapid surveillance of tissue pathology.

Two primary challenges were identified in the work in aim 1. First, while SCA can be used to isolate features, such as APFs, from heterogeneous images, its performance is limited by the contrast between APFs and the background. Second, while it is feasible to create mosaics by scanning a sarcoma tumor bed in a mouse, which is on the order of 3-7 mm in any one dimension, it is not feasible to evaluate an entire human surgical margin. Thus, improvements to the microscopic imaging system were made to (1) improve image contrast through rejecting out-of-focus background fluorescence and to (2) increase the field of view (FOV) while maintaining the sub-cellular resolution needed for delineation of nuclei. To address these challenges, a technique called structured illumination microscopy (SIM) was employed in which the entire FOV is illuminated with a defined spatial pattern rather than scanning a focal spot, such as in confocal microscopy.

Thus, the second aim was to improve image contrast and increase the FOV through employing wide-field, non-contact structured illumination microscopy and optimize the segmentation algorithm for new imaging modality. Both image contrast and FOV were increased through the development of a wide-field fluorescence SIM system. Clear improvement in image contrast was seen in structured illumination images compared to uniform illumination images. Additionally, the FOV is over 13X larger than the fluorescence microendoscope used in aim 1. Initial segmentation results of SIM images revealed that SCA is unable to segment large numbers of APFs in the tumor images. Because the FOV of the SIM system is over 13X larger than the FOV of the fluorescence microendoscope, dense collections of APFs commonly seen in tumor images could no longer be sparsely represented, and the fundamental sparsity assumption associated with SCA was no longer met. Thus, an algorithm called maximally stable extremal regions (MSER) was investigated as an alternative approach for APF segmentation in SIM images. MSER was able to accurately segment large numbers of APFs in SIM images of tumor tissue. In addition to optimizing MSER for SIM image segmentation, an optimal frequency of the illumination pattern used in SIM was carefully selected because the image signal to noise ratio (SNR) is dependent on the grid frequency. A grid frequency of 31.7 mm-1 led to the highest SNR and lowest percent error associated with MSER segmentation.

Once MSER was optimized for SIM image segmentation and the optimal grid frequency was selected, a quantitative model was developed to diagnose mouse sarcoma tumor margins that were imaged ex vivo with SIM. Tumor margins were stained with acridine orange (AO) in aim 2 because AO was found to stain the sarcoma tissue more brightly than acriflavine. Both acriflavine and AO are intravital dyes, which have been shown to stain nuclei, skeletal muscle, and collagenous stroma. A tissue-type classification model was developed to differentiate localized regions (75x75 µm) of tumor from skeletal muscle and adipose tissue based on the MSER segmentation output. Specifically, a logistic regression model was used to classify each localized region. The logistic regression model yielded an output in terms of probability (0-100%) that tumor was located within each 75x75 µm region. The model performance was tested using a receiver operator characteristic (ROC) curve analysis that revealed 77% sensitivity and 81% specificity. For margin classification, the whole margin image was divided into localized regions and this tissue-type classification model was applied. In a subset of 6 margins (3 negative, 3 positive), it was shown that with a tumor probability threshold of 50%, 8% of all regions from negative margins exceeded this threshold, while over 17% of all regions exceeded the threshold in the positive margins. Thus, 8% of regions in negative margins were considered false positives. These false positive regions are likely due to the high density of APFs present in normal tissues, which clearly demonstrates a challenge in implementing this automatic algorithm based on AO staining alone.

Thus, the third aim was to improve the specificity of the diagnostic model through leveraging other sources of contrast. Modifications were made to the SIM system to enable fluorescence imaging at a variety of wavelengths. Specifically, the SIM system was modified to enabling imaging of red fluorescent protein (RFP) expressing sarcomas, which were used to delineate the location of tumor cells within each image. Initial analysis of AO stained panels confirmed that there was room for improvement in tumor detection, particularly in regards to false positive regions that were negative for RFP. One approach for improving the specificity of the diagnostic model was to investigate using a fluorophore that was more specific to staining tumor. Specifically, tetracycline was selected because it appeared to specifically stain freshly excised tumor tissue in a matter of minutes, and was non-toxic and stable in solution. Results indicated that tetracycline staining has promise for increasing the specificity of tumor detection in SIM images of a preclinical sarcoma model and further investigation is warranted.

In conclusion, this work presents the development of a combination of tools that is capable of automated segmentation and quantification of micro-anatomical images of thick tissue. When compared to the fluorescence microendoscope, wide-field multispectral fluorescence SIM imaging provided improved image contrast, a larger FOV with comparable resolution, and the ability to image a variety of fluorophores. MSER was an appropriate and rapid approach to segment dense collections of APFs from wide-field SIM images. Variables that reflect the morphology of the tissue, such as the density, size, and shape of nuclei and nucleoli, can be used to automatically diagnose SIM images. The clinical utility of SIM imaging and MSER segmentation to detect microscopic residual disease has been demonstrated by imaging excised preclinical sarcoma margins. Ultimately, this work demonstrates that fluorescence imaging of tissue micro-anatomy combined with a specialized algorithm for delineation and quantification of features is a means for rapid, non-destructive and automated detection of microscopic disease, which could improve cancer management in a variety of clinical scenarios.