Enhanced phylogenetic insights into the microbiome of chronic rhinosinusitis through the novel application of long read 16S rRNA gene amplicon sequencing.

INTRODUCTION: 16S rRNA next generation sequencing (NGS) has been the de facto standard of microbiome profiling. A limitation of this technology is the inability to accurately assign taxonomy to a species order. Long read 16S sequencing platforms, including Oxford Nanopore Technologies (ONT), have the potential to overcome this limitation. The paranasal sinuses are an ideal niche to apply this technology, being a low biomass environment where bacteria are implicated in disease propagation. Characterising the microbiome to a species order may offer new pathophysiological insights. METHODOLOGY: Cohort series comparing ONT and NGS biological conclusions. Swabs obtained endoscopically from the middle meatus of 61 CRSwNP patients underwent DNA extraction, amplification and dual sequencing (Illumina Miseq (NGS) and ONT GridION). Agreement, relative abundance, prevalence, and culture correlations were compared. RESULTS: Mean microbiome agreement between sequencers was 61.4%. Mean abundance correlations were strongest at a familial/genus order and declined at a species order where NGS lacked resolution. The most significant discrepancies applied to Corynebacterium and Cutibacterium, which were estimated in lower abundance by ONT. ONT accurately identified 84.2% of cultured species, which was significantly higher than NGS. CONCLUSIONS: ONT demonstrated superior resolution and culture correlations to NGS, but underestimated core sinonasal taxa. Future application and optimisation of this technology can advance our understanding of the sinonasal microenvironment.

Low pH nasal rinse solution enhances mupirocin antimicrobial efficacy.

BACKGROUND: Chronic rhinosinusitis (CRS) is a common condition negatively impacting a patient's quality of life. It has been hypothesized that bacterial biofilms are involved in the pathogenesis of CRS due to their persistence and difficulty to eradicate with conventional antibiotic therapy. Hence, the topical delivery of antibiotics via nasal rinse solution has gained a lot of attention due to the ability to deliver higher local concentrations, with less systemic absorption and side effects. This study investigates the efficacy of mupirocin dissolved in the 3 most commonly used sinus rinses in Australia Neilmed (isotonic saline), Flo Sinus Care (sodium chloride, sodium bicarbonate, potassium chloride, glucose anhydrous and calcium lactate and Pentahydrate) and FloCRS (sodium chloride, potassium chloride and xylitol). METHODS: Planktonic and biofilm cultures of S. aureus (ATCC25923, 2 methicillin-resistant S. aureus (MRSA) (C222 and C263), and 2 methicillin-susceptible S. aureus (MSSS) (C311 and C349) clinical isolates) were treated with mupirocin dissolved in three sinus rinses (Neilmed, Flo Sinus Care and FloCRS with different pH). To establish whether pH was a significant factor in determining antibiotic activity, experiments with Flo CRS were performed both at pH 5.64 and elevated pH 7.7. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for planktonic cells. The biofilm biomass and metabolic activity were assessed by using crystal violet assay and alamarBlue assay respectively. RESULTS: The combination of mupirocin in low pH (pH 5.64) sinus rinse (FloCRS) had the highest efficacy in reducing the growth of S. aureus in both the planktonic and biofilm forms. Mupirocin diluted in FloCRS (pH 5.64) showed a significantly higher reduction in both biomass and metabolic activity than that was observed when mupirocin was diluted in Neilmed, Flo Sinus Care or FloCRS (pH 7.7). CONCLUSION: The choice of irrigant solution for topical mupirocin delivery appears to be important for antimicrobial activity. The delivery of mupirocin via low pH FloCRS could be useful in eliminating S. aureus biofilms present on the sinus mucosa of patients with CRS.

Low pH nasal rinse solution enhances mupirocin antimicrobial efficacy.

BACKGROUND: Chronic rhinosinusitis (CRS) is a common condition negatively impacting a patient's quality of life. It has been hypothesized that bacterial biofilms are involved in the pathogenesis of CRS due to their persistence and difficulty to eradicate with conventional antibiotic therapy. Hence, the topical delivery of antibiotics via nasal rinse solution has gained a lot of attention due to the ability to deliver higher local concentrations, with less systemic absorption and side effects. This study investigates the efficacy of mupirocin dissolved in the 3 most commonly used sinus rinses in Australia Neilmed (isotonic saline), Flo Sinus Care (sodium chloride, sodium bicarbonate, potassium chloride, glucose anhydrous and calcium lactate and Pentahydrate) and FloCRS (sodium chloride, potassium chloride and xylitol). METHODS: Planktonic and biofilm cultures of S. aureus (ATCC25923, 2 methicillin-resistant S. aureus (MRSA) (C222 and C263), and 2 methicillin-susceptible S. aureus (MSSS) (C311 and C349) clinical isolates) were treated with mupirocin dissolved in three sinus rinses (Neilmed, Flo Sinus Care and FloCRS with different pH). To establish whether pH was a significant factor in determining antibiotic activity, experiments with Flo CRS were performed both at pH 5.64 and elevated pH 7.7. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for planktonic cells. The biofilm biomass and metabolic activity were assessed by using crystal violet assay and alamarBlue assay respectively. RESULTS: The combination of mupirocin in low pH (pH 5.64) sinus rinse (FloCRS) had the highest efficacy in reducing the growth of S. aureus in both the planktonic and biofilm forms. Mupirocin diluted in FloCRS (pH 5.64) showed a significantly higher reduction in both biomass and metabolic activity than that was observed when mupirocin was diluted in Neilmed, Flo Sinus Care or FloCRS (pH 7.7). CONCLUSION: The choice of irrigant solution for topical mupirocin delivery appears to be important for antimicrobial activity. The delivery of mupirocin via low pH FloCRS could be useful in eliminating S. aureus biofilms present on the sinus mucosa of patients with CRS.

The clinical outcomes of medical therapies in chronic rhinosinusitis are independent of microbiomic outcomes: a double-blinded, randomised placebo-controlled trial.

BACKGROUND: Oral and topical corticosteroids, and antibiotics form the mainstay medical treatment of chronic rhinosinusitis (CRS). Clinical outcomes vary depending on the chosen therapy, resident microbiome and disease phenotype. We conducted a double- blinded, placebo-controlled Randomised Controlled Trial (RCT) to investigate effects of medical therapy on clinical outcomes and associated microbiome shifts. METHODOLOGY: Fifty eligible patients (CRS with and without polyps) were treated for 3 weeks after randomisation into 3 arms: na- mely oral prednisolone, topical budesonide irrigations and oral doxycycline; each with appropriate placebo. Clinical scoring and microbiome swabs were performed on enrolment, at treatment completion and 3-weeks post treatment completion. Microbiome analysis was performed using the llumina-MiSeq next generation sequencing platform and QIME-2 pipeline. RESULTS: Significant improvement in clinical scores was observed in prednisolone and budesonide arms at treatment completion but not with antibiotic. Sub-group analysis showed more pronounced effects in patients with polyposis. Corynebacterium and Staphylococcus species predominated, with variable bacterial relative abundance among different treatments at all time-points. The only significant microbiome finding was an increase in bacterial diversity in topical budesonide group immediately after treatment, which returned to baseline 3-weeks post treatment. CONCLUSION: Clinical improvement was significant with oral and topical steroid but not empirical antibiotic. Although there were some associated microbiome changes with the various treatments, we could not ascertain the consistency of these and whether they do have a clinical significance at all.

Zinc-depletion associates with tissue eosinophilia and collagen depletion in chronic rhinosinusitis.

BACKGROUND: Zinc plays an important role in many biological processes. Reduced zinc levels have been found in chronic rhinosinusitis (CRS) patients, however, its role in the pathophysiology of this disease remains unknown. This study examined zinc levels in the serum, mucus and tissue from CRS patients in relation to collagen content and eosinophil infiltration. The effect of zinc depletion on inflammatory cytokine production and collagen synthesis was assessed in vitro. METHODOLOGY: Zinc levels were determined in serum, mucus and tissue from controls, CRS with (CRSwNP) and without nasal polyps (CRSsNP) patients. Tissue zinc levels, collagen and inflammatory cell infiltration was examined using zinquin assays, immunofluorescence and histology on Tissue Micro Arrays. Cytokine expression and collagen synthesis was evaluated in zinc depleted primary human nasal epithelial cells (HNECs) and primary fibroblasts. RESULTS: CRSwNP patients showed reduced tissue zinc levels in correlation with a reduction in collagen content, and increased eosinophil numbers. Zinc depletion of HNECs and fibroblasts induced the production of pro-inflammatory cytokines and MUC5AC and reduced collagen secretion. CONCLUSIONS: These results suggest mucosal zinc depletion associates with tissue eosinophilia and collagen depletion in CRSwNP and induces pro-inflammatory cytokine expression and reduction of collagen synthesis in vitro.

Association between group 2 innate lymphoid cells enrichment, nasal polyps and allergy in chronic rhinosinusitis.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) were shown to be involved in the initiation and coordination of Th2-type immune responses in allergic disease animal models. Recently, ILC2s enrichment was noted in chronic rhinosinusitis (CRS) patients; however, the role of ILC2s in coordinating the Th2 response in CRS remains to be elucidated. Here, we characterize the ILC2 compartment in CRS by investigating the correlations between ILC2s, Th2 cells and Th2 cytokines expression in CRS patients. METHODS: We used flow cytometric analysis of sinonasal mucosal tissues of 29 CRS patients and 5 controls to quantify ILC2s and Th2 cells. Messenger RNA expression levels of IL-5, IL-13, IL-25, IL-33, TSLP and GATA3 were determined using qRT-PCR. RESULTS: ILC2s were significantly enriched in nasal polyps (CRSwNP) patients. Multivariate linear regression showed a significant positive association of ILC2 numbers with CRSwNP and allergic CRS and a negative association with the number of previous endoscopic sinus surgeries. Group 2 innate lymphoid cell numbers significantly correlated with Th2 cell frequencies. Messenger RNA expression levels of IL-5 and IL-13 were increased in CRSwNP compared with controls, while mRNA levels of IL-25 and GATA3 were significantly reduced. CONCLUSIONS: Our results characterize the complex interactions between ILC2s and other Th2 response elements in the context of CRS and suggest that ILC2 enrichment occurs in CRSwNP and in allergic CRS patients.

Small-colony variants and phenotype switching of intracellular Staphylococcus aureus in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) has been linked to the gram-positive bacteria Staphylococcus aureus (S. aureus) in its biofilm or intracellular forms. Recent evidence suggests that S. aureus also exists in a small-colony variant (SCV) form as a mechanism of altering its virulence capabilities. The aim of this study was to investigate the presence of SCVs in sinonasal mucosa of CRS patients and whether the phenomenon of phenotype switching can be applied to intracellular epithelial infections. METHODS: Sinonasal specimens were examined for the presence of intramucosal S. aureus and characterized to the strain level. An airway epithelial cell culture infection model was utilized to investigate whether bacteria were capable of alterations in virulence phenotype. RESULTS: Intramucosal organisms harvested from sinonasal biopsies demonstrate phenotypic growth patterns and lack of coagulase activity consistent with SCVs. Intracellular infection of airway epithelial cell cultures with S. aureus led to decreased secretion of enterotoxins and phenotypic growth alterations consistent with SCVs. CONCLUSIONS: Regulation of S. aureus virulence factors is a dynamic process, and exposure to the intracellular environment appears to provide the necessary conditions to enable these alterations in an attempt for the bacterium to survive and persist within host tissues. Further work is required to ascertain whether SCVs in CRS hold a clinically relevant pathogenic role in recalcitrant disease.

Autoimmunity and hypothyroidism in patients with uveal melanoma.

Among patients with advanced melanoma, the development of autoimmune phenomena or of hypothyroidism during therapy has been associated with a favourable outcome. The objective of this study was to determine the prevalence of autoimmunity and of hypothyroidism in the melanoma population as a whole and to determine if these disease states confer a survival advantage for patients with metastatic disease. We report our findings in the uveal melanoma population. The study population (n = 91) consisted of all patients registered at this institution with the diagnosis of uveal melanoma during a 2 year study period. Eight (8.8%) had a systemic autoimmune disease; 12 (13.2%) were hypothyroid, including 9/46 (19.6%) females. Survival of the stage 4 patients was determined from diagnosis of the primary tumour (SvDx) and from diagnosis of metastatic disease (SvMt), and was compared to that of age/sex matched stage 4 controls. For autoimmune patients versus controls, the median SvDx was 111 months vs 37 months (P = 0.2734) and the median SvMt was 17 months vs 4 months (P = 0.0887). For the hypothyroid patients versus controls, the median SvDx was 58 months vs 49 months (P = 0.5348) and the median SvMt was 4 months vs 8 months (P = 0.2437). We conclude that there is a trend toward longer survival from the date of metastasis in uveal melanoma patients with a systemic autoimmune disorder, suggesting that systemic autoimmunity may play a role in modifying the activity of established metastases. This trend is not seen among the uveal melanoma patients with hypothyroidism. The high prevalence of hypothyroidism suggests a possible molecular interaction between the two disease processes.

Retinoic acid receptor-beta as a prognostic indicator in stage I non-small-cell lung cancer.

PURPOSE: Retinoids are pivotal in the growth and differentiation of certain epithelial tissues, interacting with nuclear retinoid receptors (the retinoic acid receptors [RARs] and retinoid X receptors [RXRs]), which function as transcription factors. RAR-beta mRNA is undetectable by in situ hybridization (ISH) in 50% of non-small-cell lung cancers (NSCLC). RAR-beta may suppress tumorigenicity. Therefore, we hypothesized that loss of expression of RAR-beta gene in stage I NSCLC is a prognostic factor of a poor clinical outcome. PATIENTS AND METHODS: We retrospectively analyzed RAR-beta mRNA levels (by ISH using a digoxigenin-labeled antisense riboprobe) in specimens from 185 consecutive patients with completely resected clinical/radiographic stage I NSCLC for whom clinical follow-up data were available. RESULTS: One hundred fifty-six patients who met the criteria of pathologic stage I NSCLC and positivity for RXR-alpha mRNA (used as a control to assess RNA degradation) and who had adequate follow-up could be evaluated. RAR-beta mRNA expression was undetectable in 51 patients, weakly positive in 64 patients, and strongly positive in 41 patients. Overall survival of the 41 patients with strongly positive RAR-beta was significantly worse than for the 115 patients with weak or absent RAR-beta (P =.045). CONCLUSION: Unexpectedly, strong RAR-beta expression was associated with a significantly worse outcome of early-stage NSCLC. The mechanisms underlying this clinically and biologically important finding should be further explored.

Adenocarcinoma of the lung in young patients: the M. D. Anderson experience.

BACKGROUND: Surveillance, Epidemiology, and End Results (SEER) data for the years 1973-1992 documented that patients age < 50 years presented with more advanced disease. Because of the increase in the incidence rate of lung adenocarcinoma in the past few decades and the presentation of more advanced disease in young patients, this study was performed to determine whether differences in survival exist between younger and older patients with this disease. METHODS: The authors reviewed the experience of the University of Texas M. D. Anderson Cancer Center between 1985-1994, encompassing 157 patients age < 40 years of 4097 patients registered with adenocarcinoma of the lung. For comparison, 157 patients age > 50 years with lung adenocarcinoma were selected; these patients were matched for gender, stage of disease at presentation, and definitive therapy modality to assess survival differences more accurately. Data regarding exposure to second-hand smoke were not collected secondary to lack of documentation in the charts reviewed. RESULTS: There were no significant differences between the 2 groups with regard to the overall survival rate (P = 0.34) or time to progression (P = 0.43). Smoking status (current vs. former vs. never-smoker) was not found to be predictive of survival in either the younger group (P = 0.51) or the older group (P = 0.92). CONCLUSIONS: The data from the current study indicate that overall survival and disease free survival rates were not significantly different in these two groups. Thus, the younger patient population should be treated similarly to the older patient population. However, a surprisingly high percentage of younger patients were female (45%) and had never smoked (27%), suggesting that risk factors other than active smoking may be involved in lung carcinogenesis in these patients.

Incidence of breast carcinoma in women with thyroid carcinoma.

BACKGROUND: Breast carcinoma and differentiated thyroid carcinoma(the most common endocrine malignancy) occur predominantly in women. An association between the two tumors has been suggested by some investigators, but the potential impact of treatment of one of these diseases on the development of the other remains unclear. The authors examined the relation between the occurrence of these two tumors. METHODS: There were 41,686 patients with breast carcinoma and 3662 with thyroid carcinoma who registered at The University of Texas M. D. Anderson Cancer Center between March 1944 and April 1997. Women who received both diagnoses since 1976 were identified and incidence rates and relative risks of secondary tumor development were calculated. Surveillance, Epidemiology and End Results (SEER) program data on the age-adjusted incidences of these diseases during the same time period were used for the expected incidences in the same population. RESULTS: Among 18,931 women with a diagnosis of breast carcinoma since 1976, 11 developed differentiated thyroid carcinoma > or = 2 years after the diagnosis of breast carcinoma. These breast carcinoma patients contributed 129,336 person-years of follow-up; the observed incidence of thyroid carcinoma in this group was not different from that in a similar age group of women in the SEER database. Among 1013 women with a diagnosis of thyroid carcinoma since 1976, 24 developed breast carcinoma > or = 2 years after the diagnosis of thyroid carcinoma. These thyroid carcinoma patients contributed 8380 person-years of follow-up; the observed incidence of breast carcinoma in women ages 40-49 years was significantly higher than the expected incidence for women in the same age group in the SEER database. CONCLUSIONS: Breast carcinoma developing after thyroid carcinoma was diagnosed more frequently than expected in young adult women seen at the study institution since 1976. This potential association and plausible mechanisms of breast carcinoma development after thyroid carcinoma should be evaluated in larger cohorts of patients.

HIV-related malignancies: community-based study using linkage of cancer registry and HIV registry data.

For people immunosuppressed by human immunodeficiency virus (HIV), we expect an increase in cancer incidence similar to that documented in transplant patients. We examined the cancer spectrum in an HIV-infected cohort, specifically malignancies not currently associated with acquired immunodeficiency syndrome (AIDS), in relation to the general population. Cancer incidence data for residents of Harris County, Texas, diagnosed between 1975 and 1994, were linked to HIV/AIDS registry data by Soundex code and date of birth to identify malignancies in an HIV-infected cohort of 14,986 persons. Incidence of cancer in this cohort was compared to the general population by standardized incidence ratio (SIR) analysis. From the HIV-infected cohort, 2289 persons (15%) were identified as having one or more malignancies, with 97% occurring in males. The linkage alone identified 29.5% of the malignancies, of which only 28.7% were diagnosed in males. Adjusting for age, HIV-infected men and women had incidences of cancer that were 16.7 [95% confidence interval (CI) 16.1-17.3] and 2.9 (95% CI 2.3-3.7) times that expected for the general population of Harris County, Texas. Besides Kaposi's sarcoma, non-Hodgkin's lymphoma, cervix cancer and brain lymphoma, non-AIDS related malignancies of Hodgkin's lymphoma, non-melanotic skin cancer in males and colon cancer in females, exhibited significant SIRs of 5.6 (95% CI 3.6-8.4), 6.9 (95% CI 4.8-9.5) and 4.0 (95% CI 1.1-10.2). Increased incidences of lung, prostate and breast malignancies were not seen in this HIV cohort. Persons infected with HIV appear to be at increased risk for the non-AIDS related malignancies, Hodgkin's lymphoma, non-melanotic skin cancer in males and colon cancer in females.

Tuberculosis in cancer patients: an update.

The demographics of tuberculosis (TB) and the therapy of malignancies have significantly changed since the last comprehensive review of TB in cancer patients. Fifty-six patients with both TB and malignancy were identified from January 1989 through December 1994 in a population of 61,931 newly registered cancer patients. The frequency of TB in cancer patients was 90 per 100,000. TB was more frequent in foreign-born patients (p < 0.001) and in racial and ethnic minorities (p < 0.001) than in non-Hispanic whites. TB developed during therapy in 48%. TB was discovered synchronously with the malignancy in 30% and in 21% occurred > or = 18 months after therapy. Pulmonary TB occurred in 50 (89%) patients and extrapulmonary TB in nine (16%) (three had both). Chest radiographic findings did not suggest TB in 20%. TB was less frequent in lung cancer (p < 0.001), head and neck cancer (p = 0.002), and solid hematologic malignancies (p < 0.001) than it had been historically, but the frequency was unchanged in acute leukemia patients (p = 0.46). TB in cancer patients occurs at a nine times greater than in the general population. It is now most frequent in leukemia patients.

Breast cancer. Is ethnicity an independent prognostic factor for survival?

BACKGROUND: A survival difference between white and black females has been reported after diagnosis and treatment for breast carcinoma. Although multivariate analyses demonstrate that the survival difference is less or nonexistent when additional prognostic factors are considered, study results have been inconsistent. The objective of this study was to examine further the role of ethnicity as an independent prognostic factor for breast carcinoma survival among white and black females treated over a 30-year period. METHODS: Among 3382 eligible white and black females who registered at M. D. Anderson Cancer Center (MDACC) from 1958 to 1987, ethnicity, age, socioeconomic status (SES), stage of disease, and treatment were examined. Data were obtained from the hospital tumor registry and survival was compared using actuarial life tables and multivariate Cox regression analyses. RESULTS: Univariate analyses demonstrated that the survival difference between white and black females was significant, with black females having a 1.63 times higher risk of mortality at 5 years compared with white females (confidence interval (CI), 1.47-1.82), with ethnicity a significant predictor of survival (P < 0.001). After controlling for SES, stage, and treatment, the relative risk was 1.12 (CI, 1.00-1.25) and ethnicity was no longer a strong predictor of survival (P = 0.048). CONCLUSIONS: In this hospital population, the white and black female survival difference, which was highly significant when only univariate analyses were considered, became marginally significant after controlling for other prognostic factors.

Comparison of MIB-1 (Ki-67) antigen and bromodeoxyuridine proliferation indices in meningiomas.

Meningiomas from 40 adult patients were labeled immunohistochemically with monoclonal antibodies to bromodeoxyuridine (BUdR) and the Ki-67 antigen, MIB-1. The meningiomas were classified as classical, or benign (n = 31); atypical (n = 4); or malignant (n = 5). Meningeal sarcomas and hemangiopericytomas were excluded. The patient population consisted of 26 women and 14 men, ranging in age from 26 to 75 years. BUdR proliferation indices ranged from 0% to 5.8%, measurements that were expectedly lower than those for MIB-1, which ranged from 1.5% to 19.3%. MIB-1 proliferation indices were not significantly affected regarding steroid pretreatment or age. These results show a good correlation between the BUdR and MIB-1 proliferation markers (rs = 0.72; P < .0001), which supports the use of anti-MIB-1 as an alternative labeling tool to BUdR for the determination of the proliferation index in meningiomas, thus avoiding the administration of a potentially mutagenic drug.