Molecular strategy for the direct detection and identification of the most common fungal community in cerumen specimens by multiplex PCR.

Introduction. Otomycosis is a superficial fungal infection that is responsible for approximately 9-27 % of otitis externa. However, fungal communities in otomycosis are varied, but Aspergillus spp. and Candida spp. are the most common causes of this infection.Hypothesis Statement. The multiplex PCR assay is postulated to be able to directly detect more than one fungal genus in cerumen specimens.Aim. This study aimed to develop and evaluate the role of the multiplex PCR assay in detecting the most common genus of fungi that cause otomycosis directly from the cerumen specimens.Methodology. To detect Candida and Aspergillus/Penicillium genera, three pairs of primers, including pan-fungal, pan-Candida, and pan-Aspergillus/Penicillium, were used in a multiplex PCR. In order to evaluate the performance and reproducibility of the multiplex PCR. the cerumen of 140 patients suspected of otomycosis were investigated.Results. Pan-Candida and pan-Aspergillus/Penicillium primers were designed to amplify the ITS1-5.8S-ITS2 region and the ß-tubulin gene, respectively. In the multiplex PCR assay, 64 (47.40 %) and 118 (87.40 %) specimens were positive with pan-Candida and pan-Aspergillus/Penicillium primers, respectively. Double amplicon bands of Candida and Aspergillus were obtained in 51 (37.77 %) specimens. In the culture method, yeast (n=18, 13.33 %) and mould (n=117, 86.66 %) were isolated from 135 cerumen specimens. The sensitivity, specificity, and positive and negative predictive values of the multiplex PCR assays using culture method results as the gold standard were determined to be 94, 33, 97, and 22 %, respectively.Conclusion. In our study, multiplex PCR assays enabled simultaneous detection of two common genera of the causative agent of otomycosis in a cerumen specimen. Regarding the high sensitivity of the first step of the multiplex PCR assay, this assay may be used for the direct detection of Candida and Aspergillus genera in other clinical specimens.

Robotic intracorporeal orthotopic neobladder in the supine Trendelenburg position: a stepwise approach.

Robotic radical cystectomy with urinary diversion has become increasingly utilized for the surgical management of bladder cancer. Orthotopic neobladder reconstruction is still performed worldwide primarily via an extracorporeal approach because of the difficulty associated with robotic intracorporeal reconstruction. The objective of this article is to demonstrate a stepwise approach for robotic intracorporeal neobladder in a standardized manner that adheres to the principles of open surgery.

Overactive bladder and urgency urinary incontinence in men undergoing artificial urinary sphincter placement.

PURPOSE: To assess persistent and de novo rates of overactive bladder (OAB) and urgency urinary incontinence (UUI) in patients with incontinence after prostate treatment (IPT) focusing on differences between surgical intervention vs radiation. METHODS: We performed a retrospective review of 79 patients who underwent primary artificial urinary sphincter (AUS) placement and activation from a single surgeon between February 2012 and November 2017. Four patients with neurogenic bladder were excluded and two with insufficient follow-up. The primary outcome measures were persistent OAB, persistent UUI, and pad usage before and after AUS placement. RESULTS: After activation of the AUS, 67% of non-radiated patients had resolution of urgency incontinence vs only 31% of the radiated patients (P = .096). After activation of the AUS, resolution of OAB symptoms was more common in the non-radiated group. We found 53% of the non-radiated group vs only 22% of the radiated group had resolution of their urinary urgency (P = .045). Previous history of radiation was a risk factor for OAB after implantation of AUS (odds ratio [OR], 3.63; P = .010). Postoperative oral medical pharmacotherapy for OAB was higher in those with previous radiation vs those without prior radiation (66.7% vs 25.7%, P = .001). A history of OAB or UUI did not affect social continence after AUS placement. CONCLUSION: Radiation is a risk for continued OAB after AUS activation. Appropriate counseling is necessary pre- and postoperatively to manage patient expectations and provide additional medical therapies. Mixed urinary incontinence or OAB symptoms should not exclude patients from undergoing AUS placement.

Pediatric Kidney Transplantation Outcomes in Children with Primary Urological Abnormalities Versus Nonurological Abnormalities: Long-Term Results.

PURPOSE: We assessed renal function, graft survival rates and the risk of graft loss in children based on etiology with a focus on differences between urological causes from congenital anomalies of the kidney and urinary tract vs other causes of end stage kidney disease. MATERIALS AND METHODS: A retrospective chart review was performed including patients younger than 18 years who underwent kidney transplantation at our institution from December 1984 to November 2010 with the last followup recorded in March 2018. Patient clinical characteristics, demographics and end stage kidney disease etiology were recorded. Patients were divided into the 2 groups of urological (congenital anomalies of the kidney and urinary tract) vs nonurological based on end stage kidney disease etiology, and survival analysis was performed. RESULTS: Of 112 kidney transplant cases 90 (80.4%) were associated with nonurological causes and 22 (19.6%) with urological causes. Median (IQR) patient age at transplantation was 12 (7-15) years. Median graft survival time was not statistically different according to end stage kidney disease etiology (nonurological 12 years 95% CI 10.01-13.99 vs urological 16 years 95% CI 7.59-24.41, p=0.532). There was a significant risk of graft loss in patients with urinary tract infections after transplantation (HR 3.15, 95% CI 1.59-6.25, p=0.001). CONCLUSIONS: Children requiring transplantation due to urological causes have no disadvantage in graft survival compared to children with end stage kidney disease with other causes. Patients with urinary tract infection after transplantation had a higher rate of graft loss.

Prenatal renal parenchymal area as a predictor of early end-stage renal disease in children with vesicoamniotic shunting for lower urinary tract obstruction.

BACKGROUND: Vesicoamniotic shunting (VAS) and other bladder drainage techniques for fetal lower urinary tract obstruction (LUTO) have been proven to ameliorate pulmonary hypoplasia and increase survival in patients with an initial poor prognosis. Currently there are limited prognostic tools available during gestation to evaluate and predict postnatal renal function. OBJECTIVE: The aim was to describe the prenatal growth of the renal parenchymal area (RPA) in patients with LUTO and determine its application as a predictor of renal function at one year of life. STUDY DESIGN: The study population comprised a retrospective cohort of all infants who survived the fetal VAS to birth. Renal growth and size were measured using imageJ software to calculate the RPA in sequential prenatal ultrasounds. The parenchymal area was measured from the image of each kidney with the greatest longitudinal length. These measurements were further correlated and analyzed as a predictor of end-stage renal disease (ESRD) within the first year of life. RESULTS: Etiologies of LUTO in the 15 male fetuses included eight posterior urethral valves, four Eagle-Barrett/prune belly syndrome, two urethral atresia, and one megacystis microcolon intestinal hypoperistalsis syndrome. All patients had patent shunts, in place, at birth. Furthermore, ultrasonographic parameters such as oligohydramnios, keyhole sign, and bladder wall thickness showed no statistical difference between groups. Renal parenchymal growth correlated with postnatal renal function in both the ESRD (r = 0.409, p = 0.018) and the non-ESRD (r = 0.657, p < 0.001) groups. Most notably, RPA during the 3rd trimester predicted ESRD with the best cut-off point determined to be 8 cm2 (sensitivity, 0.714; specificity, 0.882; and positive likelihood ratio, 6.071) (Table). DISCUSSION: Despite definitive VAS for LUTO, postnatal morbidity and mortality remain high, emphasizing the role of renal dysplasia in postnatal renal failure, in spite of urinary diversion. Renal growth statistically differs between groups in the 3rd trimester of gestation; RPA development appears stagnant in patients that developed ESRD within the first year of life. In contrast, patients that did not develop ESRD continued to have renal parenchymal growth in a linear fashion. This suggests that prenatal RPA may be predictive of postnatal ESRD. CONCLUSIONS: RPA measurement during the prenatal period could play an important role as a non-invasive tool to predict postnatal renal function and to anticipate postnatal clinical interventions.

Decreased Sensitization to Aeroallergens among Southwestern Iranian Male Farmers.

Farmers are usually exposed to various inhaled allergens like pollens, mites, molds, and animal dander in their working environment which may lead to allergic rhinitis, asthma and urticaria. The purpose of this study was to identify sensitization to various aeroallergens in farmers and their occupational allergy symptoms. This cross sectional study included 103 male farmers and 100 non-farmer healthy controls. The work-related symptoms of farmers were recorded with a questionnaire. Spirometry and skin prick tests with 15 commercial allergen extracts were performed in both farmers and controls. The rate of sensitization to at least one of the applied aeroallergens was 47.6% in farmers compared to 65% in the control group (OR=0.48; CI 95%, 1.08 to 2.07) according to skin prick tests, after adjusting for age. Occupational allergy symptoms were reported by 54.3% farmers. Mean FEV1/FVC was significantly lower in farmers than in controls (p<0.001). The results of this study showed that farmers had no increased risk of sensitization to aeroallergens. Sensitization to pollens was more prevalent than to mites among the farmers in our study and smoking was an important predisposing factor in farmers who suffered from occupational allergy symptoms.

Across-shift changes of exhaled nitric oxide and spirometric indices among cotton textile workers.

OBJECTIVES: For the purpose of evaluation of exhaled NO as an index of airway inflammation, we assessed changes in fractional exhaled NO (FeNO) across a work shift and its relationship with respiratory complaints. MATERIAL AND METHODS: Chronic and work-aggravated respiratory complaints were assessed using a questionnaire in 89 male textile workers. FeNO and spirometry were performed before and after a work shift and all the changes were registered. RESULTS: A significant increase in FeNO after a work shift was observed. Post-shift FeNO was significantly higher among the subjects with chronic respiratory complaints. There was an obvious decrease in FVC, and FEV1 after a work shift; however, we couldn't find a significant relationship between changes in respiratory parameters and concentration of inhalable dusts. CONCLUSIONS: FeNO increase after a work shift along with pulmonary function decrement and higher post-shift FeNO among subjects with respiratory complaints makes across-shift FeNO a non-invasive test for assessment of airway hyper-responsiveness in textile workers.

UTIs in patients with neurogenic bladder.

Urinary tract infections (UTI) remain one of the most prevalent and frustrating morbidities for neurogenic bladder patients, and death attributed to urosepsis in the spinal cord injury (SCI) patient is higher when compared to the general population. Risk factors include urinary stasis, high bladder pressures, bladder stones, and catheter use. While classic symptoms of UTI include dysuria, increased frequency and urgency, neurogenic bladder patients present differently with increased spasticity, autonomic dysreflexia, urinary incontinence, and vague pains. Multiple modalities have been assessed for prevention including catheter type, oral supplements, bladder irrigation, detrusor injections and prophylactic antimicrobials. Of these, bladder inoculation with E. coli HU2117, irrigation with iAluRil(®), detrusor injections, and weekly prophylaxis with alternating antibiotics appear to have a positive reduction in UTI but require further study. Ultimately, treatment for symptomatic UTI should account for the varied flora and possible antibiotic resistances including relying on urine cultures to guide antibiotic therapy.

The association between exhaled nitric oxide and sleep apnea: the role of BMI.

BACKGROUND: Obstructive sleep apnea syndrome is associated with airway inflammation. Measurement of exhaled nitric oxide is a non-invasive method for evaluation of airway diseases. It seems that obesity is an exacerbating factor for airway inflammation. We aimed to evaluate the changes of exhaled nitric oxide after sleep in patients suffering from OSA regarding BMI. METHOD: In 54 patients referred for polysomnography, exhaled nitric oxide measurements were performed before and after sleep. Subjects were divided into three categories: normal, obese with sleep apnea and non-obese, based on polysomnographic recordings and BMI. RESULTS: 47 subjects had abnormal apnea/hypopnea index (AHI mean = 39.7) and 7 were normal regarding AHI (AHI mean = 3.0). BMI was significantly correlated to AHI, number of desaturations and hypoxia. Among those with apnea, 31 subjects were obese and 16 were non-obese. Exhaled nitric oxide levels in normal and OSA subjects showed no significant change, but a significant increase was found in obese patients with apnea (14.7 pre-sleep mean, 20.0 post-sleep mean). CONCLUSIONS: Obesity is an effective factor in the inflammation of airways in patients with obstructive apnea.

Succinate dehydrogenase mutation underlies global epigenomic divergence in gastrointestinal stromal tumor.

Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT, or, alternatively, manifest loss-of-function defects in the mitochondrial succinate dehydrogenase (SDH) complex, a component of the Krebs cycle and electron transport chain. We have uncovered a striking divergence between the DNA methylation profiles of SDH-deficient GIST (n = 24) versus KIT tyrosine kinase pathway-mutated GIST (n = 39). Infinium 450K methylation array analysis of formalin-fixed paraffin-embedded tissues disclosed an order of magnitude greater genomic hypermethylation relative to SDH-deficient GIST versus the KIT-mutant group (84.9 K vs. 8.4 K targets). Epigenomic divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (n = 29), a developmentally distinct SDH-deficient tumor system. Comparison of SDH-mutant GIST with isocitrate dehydrogenase-mutant glioma, another Krebs cycle-defective tumor type, revealed comparable measures of global hypo- and hypermethylation. These data expose a vital connection between succinate metabolism and genomic DNA methylation during tumorigenesis, and generally implicate the mitochondrial Krebs cycle in nuclear epigenomic maintenance.

Overexpression of insulin-like growth factor 1 receptor and frequent mutational inactivation of SDHA in wild-type SDHB-negative gastrointestinal stromal tumors.

Approximately 15% of gastrointestinal stromal tumors (GISTs) in adults and 85% in children lack mutations in KIT and PDGFRA and are known as wild-type GISTs. Wild-type GISTs from adults and children express high levels of insulin-like growth factor 1 receptor (IGF1R) and exhibit stable genomes compared to mutant GISTs. Pediatric wild-type GISTs, GISTs from the multitumor Carney-Stratakis syndrome, and the Carney triad share other clinicopathological properties (e.g., early-onset, multifocal GISTs with epitheliod cell morphology), suggesting a common etiology. Carney-Stratakis is an inherited association of GIST and paragangliomas caused by germline mutations in succinate dehydrogenase (SDH) genes. The connection between defective cellular respiration and GIST pathology has been strengthened by the utilization of SDHB immunohistochemistry to identify SDH deficiency in pediatric GISTs, syndromic GISTs, and some adult wild-type GISTs. SDHB and IGF1R expression was examined in 12 wild-type and 12 mutant GIST cases. Wild-type GISTs were screened for coding-region alterations in SDH genes and for chromosomal aberrations using genome-wide single-nucleotide polymorphism and MIP arrays. SDHB-deficiency, identified in 11/12 wild-type GIST cases, was tightly associated with overexpression of IGF1R protein and transcript. Biallelic inactivation of the SDHA gene was a surprisingly frequent event, identified in 5 of 11 SDHB-negative cases, generally due to germline point mutations accompanied by somatic SDHA allelic losses. As a novel finding, inactivation of the SDHC gene from a combination of a heterozygous coding-region mutation and hypermethylation of the wild-type allele was found in one SDHB-negative case.

Molecular inversion probe analysis detects novel copy number alterations in Ewing sarcoma.

Ewing sarcoma (ES) is the second most common bone tumor in children and young adults, with dismal outcomes for metastatic and relapsed disease. To better understand the molecular pathogenesis of ES and to identify new prognostic markers, we used molecular inversion probes (MIPs) to evaluate copy number alterations (CNAs) and loss of heterozygosity (LOH) in formalin-fixed paraffin-embedded (FFPE) samples, which included 40 ES primary tumors and 12 ES metastatic lesions. CNAs were correlated with clinical features and outcome, and validated by immunohistochemistry (IHC). We identified previously reported CNAs, in addition to SMARCB1 (INI1/SNF5) homozygous loss and copy neutral LOH. IHC confirmed SMARCB1 protein loss in 7-10% of clinically diagnosed ES tumors in three separate cohorts (University of Utah [N = 40], Children's Oncology Group [N = 31], and University of Michigan [N = 55]). A multifactor copy number (MCN)-index was highly predictive of overall survival (39% vs. 100%, P < 0.001). We also identified RELN gene deletions unique to 25% of ES metastatic samples. In summary, we identified both known and novel CNAs using MIP technology for the first time in FFPE samples from patients with ES. CNAs detected by microarray correlate with outcome and may be useful for risk stratification in future clinical trials.

Differentiation of malignant melanoma from benign nevus using a novel genomic microarray with low specimen requirements.

CONTEXT: Histologic examination of clinically suspicious melanocytic lesions is very sensitive and specific for the detection of malignant melanoma. Yet, the malignant potential of a small percentage of melanocytic lesions remains histologically uncertain. Molecular testing offers the potential to detect the genetic alterations that lead to malignant behavior without overt histologic evidence of malignancy. OBJECTIVE: To differentiate benign melanocytic nevi from malignant melanoma and to predict the clinical course of melanocytic lesions with ambiguous histology using a novel genomic microarray. DESIGN: We applied a newly developed single-nucleotide polymorphism genomic microarray to formalin-fixed, paraffin-embedded melanocytic lesions to differentiate benign nevi (n  =  23) from malignant melanoma (n  =  30) and to predict the clinical course of a set of histologically ambiguous melanocytic lesions (n  =  11). RESULTS: For cases with unambiguous histology, there was excellent sensitivity and specificity for identifying malignant melanoma with this genomic microarray (89% sensitivity, 100% specificity). For cases with ambiguous histology, the performance of this genomic microarray was less impressive. CONCLUSIONS: Without microdissection and with quantities of DNA one-tenth what is required for more commonly used microarrays, this microarray can differentiate between malignant melanoma and benign melanocytic nevi. For histologically ambiguous lesions, longer clinical follow-up is needed to confidently determine the sensitivity and specificity of this microarray. Some of the previous technical hurdles to the clinical application of genomic microarray technology are being overcome, and the advantages over targeted fluorescence in situ hybridization assays currently in clinical use are becoming apparent.

Melanoma mimic: a case of multiple pagetoid Spitz nevi.

BACKGROUND: Differentiating Spitz nevi from melanoma can be difficult. Pagetoid spread of melanocytes is among the features making diagnosis difficult. Rare reports of isolated pagetoid Spitz nevi exist. OBSERVATIONS: We present a unique case of multiple pagetoid Spitz nevi initially diagnosed as multiple in situ melanomas. Germline karyotyping, CDK4 and CDKN2A sequencing, and comparative genomic hybridization of HRAS, BRAF, KRAS, RAF1, CDKN2A, Rb1, MAP2K1, MAP2K2, PTEN, and PTPN11 genes did not identify mutations in this case. Germline and somatic sequencing of BRAF exon 15 revealed no mutations at V600D/E/K. In addition, single-nucleotide polymorphism microarray analysis (330K) on lesional and normal skin revealed no genome-wide copy number changes or loss of heterozygosity. CONCLUSIONS: Clinicians should be aware of the occurrence of multiple pagetoid Spitz nevi to avoid morbidity associated with the misdiagnosis of multiple melanomas. The genetic mechanisms of pagetoid spread of melanocytes are not fully understood.

FOXL2 mutation and large-scale genomic imbalances in adult granulosa cell tumors of the ovary.

Adult granulosa cell tumors (AGCTs) are a rare class of ovarian tumors with recurrent cytogenetic abnormalities including trisomy 12, trisomy 14, monosomy 16/deletion 16q, and monosomy 22. Over 90% contain a missense point mutation (C134W) in the FOXL2 gene at 3q22.3. The relationship between FOXL2 mutation and cytogenetic abnormalities is unclear, although both are presumably early events in tumorigenesis. In addition, FOXL2 C134W mutant allele imbalance has been noted in a minority of AGCTs, but the mechanism for allelic imbalance has not yet been described. We used a microarray platform designed for formalin-fixed, paraffin-embedded (FFPE) tissue specimens, the Affymetrix OncoScan FFPE Express 330K Molecular Inversion Probe (MIP) array, to explore the correlation between genomic imbalances detected by microarray and FOXL2 mutation status detected by pyrosequencing in a series of 21 archived AGCTs. Tumors were characterized by histopathologic features, stage, and alpha-inhibin expression by immunohistochemistry. All tumors were positive for inhibin, and 18/21 tumors contained a FOXL2 mutation. The most common genomic imbalances were a gain of 14q, a loss of 16q, and a loss of 22q. Three tumors showed evidence of FOXL2 mutant allele imbalance by pyrosequencing; microarray revealed a 32.5 Mb deletion encompassing FOXL2 in 1 case and a 70.9 Mb stretch of homozygosity encompassing FOXL2 in the other case. The third case, with a FOXL2 mutant allele imbalance, showed a diminished mutant allele population (32%) despite high estimated tumor content (>90%), suggesting tumor heterogeneity for the mutation. This study provides the first correlation of FOXL2 mutation status and genomic imbalances in AGCTs, and it further elucidates the mechanisms for mutant allele imbalance in cancer.

Genome wide copy number analysis of paediatric Burkitt lymphoma using formalin-fixed tissues reveals a subset with gain of chromosome 13q and corresponding miRNA over expression.

The majority of paediatric Burkitt lymphoma (pBL) patients that relapse will die of disease, but markers for this high-risk subset are unknown. MYC translocations characterize pBL, but additional genetic changes may relate to prognosis and serve as potential biomarkers. We utilized a molecular inversion probe single nucleotide polymorphism assay to perform high resolution, genome-wide copy number analysis on archival formalin-fixed, paraffin-embedded pBL and germline tissues. We identified copy number abnormalities (CNAs) in 18/28 patients (64%) with a total of 62 CNAs that included 32 gains and 30 copy number losses. We identified seven recurrent CNAs including 1q gain (7/28, 25%), 13q gain (3/28, 11%), and 17p loss (4/28, 14%). The minimum common amplified region on 13q was at 13q31 and included the MIR17HG (MIR17-92) locus. Samples with this gain had higher levels of MIR17 RNA and showed a tendency for early relapse. Tumour-specific uniparental disomy was identified in 32% of cases and usually was recurrent. These results demonstrate that high-resolution copy number analysis can be performed on archival lymphoma tissue specimens, which has significance for the study of rare diseases.

Copy Number Alterations and Methylation in Ewing's Sarcoma.

Ewing's sarcoma is the second most common bone malignancy affecting children and young adults. The prognosis is especially poor in metastatic or relapsed disease. The cell of origin remains elusive, but the EWS-FLI1 fusion oncoprotein is present in the majority of cases. The understanding of the molecular basis of Ewing's sarcoma continues to progress slowly. EWS-FLI1 affects gene expression, but other factors must also be at work such as mutations, gene copy number alterations, and promoter methylation. This paper explores in depth two molecular aspects of Ewing's sarcoma: copy number alterations (CNAs) and methylation. While CNAs consistently have been reported in Ewing's sarcoma, their clinical significance has been variable, most likely due to small sample size and tumor heterogeneity. Methylation is thought to be important in oncogenesis and balanced karyotype cancers such as Ewing's, yet it has received only minimal attention in prior studies. Future CNA and methylation studies will help to understand the molecular basis of this disease.