The Role of Histological Examination of Nail Clipping in the Diagnosis of Onychomycosis.

BACKGROUND: Diagnosis of onychomycosis is based on potassium hydroxide (KOH), direct smear, culture, and polymerase chain reaction. Nail clippings are rarely used as a diagnostic tool. OBJECTIVES: To evaluate nail clippings for the diagnosis of onychomycosis and to compare it to KOH smears. METHODS: Nail clipping specimens of 39 patients were collected: 34 with onychomycosis proved by positive culture and 5 from normal nails. The specimens were submitted to histological processing and then stained with periodic acid-Schiff (PAS) and Grocott-Gomori's methenamine silver (GMS) stains. For each nail, KOH smear was also performed. Two pathologists who had no information on the KOH smear and the culture results evaluated the nail clipping histology for the presence of fungal element. Their assessment was compared to the KOH smear and culture results. RESULTS: Of the 34 specimens that had positive culture, 25 were dermatophytes, 5 were molds, and 4 were candida. Clipping specimens were positive in 30 cases (88%): 23/25 dermatophyte, 4/5 molds, and 3/4 candida. Pathologists were able to classify the pathogens into dermatophytes and non-dermatophytes based on the morphology. PAS stain results were the same as GMS in evaluation of the nail specimen. KOH smear was positive in 29 nails (85%): 20/25 dermatophytes, all 5 molds, and 4 candida. In all five nails where the culture was negative, both clipping and KOH smear did not show fungal elements. CONCLUSIONS: Nail clippings can serve as a rapid, inexpensive, and reliable method for evaluation of onychomycosis, comparable to KOH smear, with the advantage of pathogen group identification.

Endometrial glycogen metabolism during early pregnancy in mice.

Glucose is critical during early pregnancy. The uterus can store glucose as glycogen but uterine glycogen metabolism is poorly understood. This study analyzed glycogen storage and localization of glycogen metabolizing enzymes from proestrus until implantation in the murine uterus. Quantification of diastase-labile periodic acid-Schiff (PAS) staining showed glycogen in the glandular epithelium decreased 71.4% at 1.5 days postcoitum (DPC) and 62.13% at DPC 3.5 compared to proestrus. In the luminal epithelium, glycogen was the highest at proestrus, decreased 46.2% at DPC 1.5 and 63.2% at DPC 3.5. Immunostaining showed that before implantation, glycogen metabolizing enzymes were primarily localized to the glandular and luminal epithelium. Stromal glycogen was low from proestrus to DPC 3.5. However, at the DPC 5.5 implantation sites, stromal glycogen levels increased sevenfold. Similarly, artificial decidualization resulted in a fivefold increase in glycogen levels. In both models, decidualization increased expression of glycogen synthase as determine by immunohistochemistry and western blot. In conclusion, glycogen levels decreased in the uterine epithelium before implantation, indicating that it could be used to support preimplantation embryos. Decidualization resulted in a dramatic increase in stromal glycogen levels, suggesting it may have an important, but yet undefined, role in pregnancy.

PodoSighter: A Cloud-Based Tool for Label-Free Podocyte Detection in Kidney Whole-Slide Images.

BACKGROUND: Podocyte depletion precedes progressive glomerular damage in several kidney diseases. However, the current standard of visual detection and quantification of podocyte nuclei from brightfield microscopy images is laborious and imprecise. METHODS: We have developed PodoSighter, an online cloud-based tool, to automatically identify and quantify podocyte nuclei from giga-pixel brightfield whole-slide images (WSIs) using deep learning. Ground-truth to train the tool used immunohistochemically or immunofluorescence-labeled images from a multi-institutional cohort of 122 histologic sections from mouse, rat, and human kidneys. To demonstrate the generalizability of our tool in investigating podocyte loss in clinically relevant samples, we tested it in rodent models of glomerular diseases, including diabetic kidney disease, crescentic GN, and dose-dependent direct podocyte toxicity and depletion, and in human biopsies from steroid-resistant nephrotic syndrome and from human autopsy tissues. RESULTS: The optimal model yielded high sensitivity/specificity of 0.80/0.80, 0.81/0.86, and 0.80/0.91, in mouse, rat, and human images, respectively, from periodic acid-Schiff-stained WSIs. Furthermore, the podocyte nuclear morphometrics extracted using PodoSighter were informative in identifying diseased glomeruli. We have made PodoSighter freely available to the general public as turnkey plugins in a cloud-based web application for end users. CONCLUSIONS: Our study demonstrates an automated computational approach to detect and quantify podocyte nuclei in standard histologically stained WSIs, facilitating podocyte research, and enabling possible future clinical applications.

Thickening of the basement membrane as a diagnostic sign of mycosis fungoides.

The epidermal basement membrane (BM) is readily identified on skin biopsy specimens stained with periodic acid-Schiff (PAS) and PAS with diastase (PAS-D). Thickening of BM can be evidenced in several inflammatory and tumoral conditions. We noticed that most of our biopsy specimens of mycosis fungoides (MF) showed thickening of the BM. We decided to retrospectively study BM thickness in 27 biopsy specimens of MF and compare them with 27 cutaneous biopsy specimens of inflammatory diseases. We studied PAS and PAS-D stains in all cases and we measured BM thickness with an ocular micrometer. Cases were scored in a four-tiered system: 0: no detectable staining; 1+ (mild: < 5 µm); 2+ (moderate: 5-9 µm); and 3+ (prominent: >9 µm). The difference between both groups (MF vs controls) was highly significant for BM thickness values by both one- and two-tailed t tests (P < 0.0006). While only 3 biopsy specimens from the controls (11.11%) showed areas of 3+ thickening, 12 biopsy specimens of MF (44.44%) showed areas of 3+ thickening, and most cases showed diffuse, at least 2+ thickening, while the controls showed more segmental, mostly 1 or 2+ staining. We conclude that thickening of BM can be useful in the differential diagnosis with inflammatory conditions.

Histopathological Findings in Nail Clippings With Periodic Acid-Schiff-Positive Fungi.

BACKGROUND: Periodic acid-Schiff (PAS) staining of nail clippings is an adjunct diagnostic tool for onychomycosis. OBJECTIVE: To detect histopathological findings as clues to the presence of PAS-positive (+) fungal elements in nail clippings. METHODS: Four hundred sixteen consecutive nail clippings suspected of onychomycosis were stained with hematoxylin and eosin, and with PAS stains. All cases were studied histopathologically. The clinical files of the cases with neutrophils were reviewed. RESULTS: PAS+ staining for fungi were demonstrated in 159 (38%) of the nail clippings. Neutrophils, parakeratosis, plasma globules, and bacteria were observed in 43 (27%), 108 (67%), 80 (50%), and 80 (50%) of the PAS+ cases, respectively, and in 17 (6%), 109 (41%), 84 (32%) and 140 (54%) of the PAS- cases, respectively (P < 0.01). Neutrophils showed by far the highest specificity (93%), although with low sensitivity (27%) for the presence of PAS+ fungi. Among the 43 PAS+ and 17 PAS- specimens with neutrophils, only 1 (2.3%) and 3 (17%) had overt psoriasis, respectively. CONCLUSION: Neutrophils in nail clippings may serve as a clue for onychomycosis. PAS staining with neutrophils is not necessarily associated with psoriasis.

CXCR5/NRF2 double knockout mice develop retinal degeneration phenotype at early adult age.

The objective of this study is to characterize the retinal degeneration (RD) phenotype of CXCR5/NRF2 double knockout (DKO) mice at the early adult age. CXCR5 KO mice and NRF2 KO mice were bred to create CXCR5/NRF2 DKO mice. The assessment of RD features included fundus and optical coherence tomography (OCT) imaging, periodic acid-Schiff (PAS), and immunofluorescence staining of retinal pigment epithelium (RPE)-choroid flatmounts. Stained samples were imaged with fluorescent microscopy, and Western blots were used to monitor protein expression changes. The staining of cleaved caspase-3 and PNA-lectin was performed to assess the presence of photoreceptor cell apoptosis. Quantification and statistical analyses were performed with Image J and Graphpad software. The young adult (2-6 months) DKO mice exhibited increased hypopigmented spots on fundus and sub-RPE abnormalities on OCT as compared to the CXCR5-KO mice, and C57BL6 WT controls. PAS-stained sections demonstrated aberrant RPE/sub-RPE depositions. The DKO mice had increased sub-RPE depositions of IgG and AMD-associated proteins (ß-amyloid, Apolipoprotein-E, C5b-9, and αB-crystallin). The protein expression of AMD-associated proteins and microglia marker (TMEM119) were upregulated at the RPE/BM/choroid complex of DKO mice. The adult DKO mice underwent photoreceptor cell apoptosis compared to the single CXCR5 and NRF2 KO and the WT mice at an early adult age. Mechanistically increased expression of CXCL13 and N-cadherin was observed as a sign of epithelial-mesenchymal transition. The data suggest that the CXCR5/NRF2-DKO mice develop RD characteristics at an early age and may serve as a valuable animal model of RD.

PAS stain based histological classification and severity grading of toenail onychomycosis.

Onychomycosis is a common world-wide health issue. Accurate detection is essential for treatment. Multiple studies have shown that PAS-stain based histological visualization of fungal elements is superior to either direct microscopy with 20% potassium hydroxide, or fungal culture. However, PAS stain based histological classification and severity grading of onychomycosis are lacking in the literature. Here we reported a fungal detection rate of 47.87% based on an analysis of 13,805 toenails processed for H&E and PAS stains over a three year period. Based on the analysis of fungal density, distribution and infiltrating depth level in 858 PAS-positive toenails, we created a novel PAS stain based histological classification system to classify onychomycosis as occult onychomycosis (OO), focal or diffuse subungual onychomycosis (FSO or DSO), focal or diffuse plate onychomycosis (FPO or DPO), focal or diffuse subungual and plate onychomycosis (FSPO or DSPO) and superficial onychomycosis (SO). The severities of OO, FSO and FPO were graded as mild, DSO and DPO as moderate, FSPO and DSPO as severe infections, which revealed that more than 75% PAS positive toenails were severe infections. Evaluation of 97 paired toenails biopsied pre- and post-treatment from 47 patients demonstrated that the severity of infection was significantly reduced from severe to mild and moderate levels. These data indicate that the current histological classification evaluates not only the severity of the fungal infection but also the response to treatment. We further propose a guideline for treatment of onychomycosis based on the histological classification and severity.

PAS and GMS utility in dermatopathology: Review of the current medical literature.

The American Society of Dermatopathology has established an Appropriate Use Criteria (AUC) Committee with the intention of establishing evidence-based recommendations regarding the appropriateness of various ancillary tests commonly utilized by dermatopathologists. Periodic acid Schiff (PAS) and Grocott (or Gomori) methenamine silver (GMS) stains represent some of the most commonly employed ancillary tests in dermatopathology. The utility of these tests was targeted for evaluation by the AUC. This literature review represents a comprehensive evaluation of available evidence for the utility of PAS and/or GMS staining of skin and nail biopsies. In concert with expert opinion, these data will be incorporated into future recommendations by the AUC for PAS and GMS staining in routine dermatopathology practice.

Effects of various fixatives and temperature on the quality of glycogen demonstration in the brain and liver tissues.

The visualization of glycogen deposits in cells and tissues is important for studying glycogen metabolism as well as diagnosis of glycogen storage diseases. Evidence suggests that the demonstration of glycogen can better be enhanced by factors such the choice of fixative and temperature during fixation. Here, we assessed efficacy of neutral buffered formalin (NBF), alcoholic formalin (AF) and paraformaldehyde (PFA) at 4 °C, 37 °C and 40 °C using Periodic Acid Schiff's staining method. Each liver specimen was fixed in NBF and AF while the brain tissues were fixed in NBF, AF and PFA. We found that there was a better PAS staining intensity with the liver tissues fixed in AF compared with NBF. Also, there was no difference in the quality of the staining for tissues fixed in AF at 37 °C, 4 °C and 40 °C, but fixation with NBF at 4 °C gave the best staining quality when compared with 40 °C and 37 °C. Furthermore, hippocampal tissues fixed in AF showed better quality of PAS staining compared with NBF and PFA. A significant increase in staining intensity was observed for PFA when compared with NBF. Superior staining intensity for PAS was observed at 4 °C for hippocampal tissues fixed with NBF, AF and PFA. Taken together our results show that AF at a temperature of 4 °C gave the best result. Hence, glycogen demonstration can better be enhanced by the choice of fixative and temperature during fixation.

Application of fungal fluorescent staining in oral candidiasis: diagnostic analysis of 228 specimens.

BACKGROUND: Several conventional methods, including fungal culture and periodic acid-Schiff (PAS) reagent staining, have been used to diagnose oral candidiasis. The aim of this study was to evaluate the efficacy of a novel method, fungal fluorescent staining, in relation to conventional protocols in the diagnosis of oral candidiasis. METHODS: We collected 106 oral swabs and 122 oral biopsy tissues from patients highly suspected with oral candidiasis. We applied fungal culture and periodic acid-Schiff reagent staining as the gold standard diagnostic tools. The efficacy of these methods in determining the presence of Candida was compared with that of fluorescent staining. RESULTS: In the majority of specimens subjected to fluorescent staining, fungal organisms were distinguished by blue fluorescence surrounding their tubular or annular shapes. The sensitivity, specificity, Youden index, positive predictive value and negative predictive value of the fluorescent staining method were 82.7, 93.5, 76.7, 96.8 and 69.1% in oral swabs and 90.0, 92.9, 82.9, 96.0 and 82.9% in oral biopsy tissues, respectively. CONCLUSIONS: Fungal fluorescent staining represents a rapid method for detection of Candida, supporting its potential utility as an effective early diagnostic tool for oral candidiasis.

Density of PAS positive patterns in uveal melanoma: Correlation with vasculogenic mimicry, gene expression class, BAP-1 expression, macrophage infiltration, and risk for metastasis.

Purpose: Periodic acid-Schiff (PAS) positive patterns of vasculogenic mimicry (VM) have been associated with poor prognosis in uveal melanoma (UM). We examined these patterns with digital image analysis and transmission electron microscopy, and correlated them with BAP-1 expression, gene expression class, macrophage infiltration, and metastatic disease in full tumor cross-sections and intratumor regions. Methods: Thirty-two enucleated eyes with UM were stained immunohistochemically (BAP-1, laminin, CD31, and CD68) and with PAS without hematoxylin counterstain. Retrospective data on gene expression class and patient survival were retrieved. Tumor sections were digitally scanned and analyzed with the QuPath Bioimage analysis software, and imaged with transmission electron microscopy. Results: The mean area proportion covered by CD31, laminin, and PAS positive patterns in tumor cross-sections was 0.9% (SD 0.6), 3.0% (SD 1.9), and 8.4% (SD 5.9), respectively. PAS density was statistically significantly greater in tumors with gene expression class 2 (p=0.02). The cumulative 5-year metastasis-free survival decreased for each quartile of increased PAS density (1.0, 0.75, 0.40, and 0.17, p=0.004). Forty percent of the tumors had heterogeneous BAP-1 expression. Intratumor regions with low BAP-1 expression were more likely to harbor VM (p<0.0001), and had statistically significantly greater PAS density (p<0.0001) and number of CD68 positive cells (p=0.01). Conclusions: PAS positive patterns in UM are composed of a mixture of blood vessels and extracellular matrix (ECM), including VM. Increased density of PAS positive patterns correlated with gene expression class and metastasis, and colocated to tumor regions with macrophage infiltration and low BAP-1 expression.

How useful is periodic acid-Schiff stain to detect fungi in biopsies from dermatoses of the palms and soles?

BACKGROUND: Periodic acid-Shiff (PAS) stain may help to diagnose fungal infection in biopsies from dermatoses of the palms and soles. It is questionable whether PAS stain should be used routinely or only when tinea is suspected clinically. METHODS: A total of 195 consecutive punch biopsies of dermatoses from the palms (90) or soles (105) were stained with PAS, regardless of the clinical differential diagnosis. RESULTS: PAS stain showed fungi in the corneal layer of 6 (3%) of the 195 biopsies. Tinea was included in the differential diagnosis in 48 cases, of which 3 (6%) were PAS positive. PAS stain was also positive in 3 (2%) of 147 cases in which tinea was not suspected clinically. All 6 PAS-positive cases were detected in reaction patterns not readily classified as particular diagnostic entities: non-inflammatory keratoderma (2, 11%), chronic lichenified dermatitis (2, 6%), spongiotic psoriasiform dermatitis (1, 2%), and spongiotic dermatitis (1, 4%). CONCLUSIONS: There is a low concordance rate between the clinical suspicion and the actual demonstration of fungi by PAS stain in dermatoses of the palms and soles. Routine PAS stains in non-suspected cases have a relatively low yield, which may be improved by limiting the PAS stain to reaction patterns not readily classified as particular diagnostic entities.

Liquid chromatography-tandem mass spectrometry is effective for analysis of ergosterol in fungal-infected nails.

BACKGROUND: Identification of onychomycosis is mainly based on clinical diagnosis with auxiliary diagnostic methods such as potassium hydroxide (KOH) microscopy, periodic acid-Schiff staining or fungal culture. However, each method is limited by its sensitivity and specificity. AIM: To develop a new test method using the common fungal end product, ergosterol, and investigate if it can be used as a new diagnostic tool. METHODS: We collected consecutive data from 20 participants with nail problems. Following clinical diagnosis, samples were taken for KOH microscopy and for mass spectrometry (MS) to check for the presence of ergosterol. RESULTS: Of the 20 cases collected, 7 were positive for fungal infection by MS. Four of these were already suspected to have onychomycosis, whereas one of the remaining three subjects was presumed to have dry nail and the other two to have onycholysis. The MS test seemed to be better at detecting combinations of nail conditions. Conversely, of the five patients clinically diagnosed as having onychomycosis, four had a positive MS result, whereas the fifth had negative results on both KOH and MS. Two other participants had a positive KOH test and were also found to have positive MS results. CONCLUSION: Detection of the presence of ergosterol by MS seems to be a useful tool for confirming onychomycosis. However, further studies are needed to verify the sensitivity and specificity of this MS method.

Characterization of N-glycosylations in Entamoeba histolytica ubiquitin.

Entamoeba histolytica harbors an extensive intracellular distribution of ubiquitin-proteasome systems important for numerous cellular processes. However, glycosylation studies of ubiquitin-proteasome components have not yet been elucidated. Here we report the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE), Nanoelectrospray Ionization-Tandem Mass Spectrometry (NSI-MS), Matrix-Assisted Laser-Desorption time-of-flight Mass Spectrometry (MALDI-TOF MS) and Gas Chromatography-Mass Spectrometry (GC-MS) analysis. To our knowledge, the data presented in this report represents the first structural glycomics analysis of E. histolytica ubiquitin, while most of the reports are performed on whole parasitic glycan profiles. The glycan profile of E. histolytica ubiquitin has high mannose N-glycan structures. The N-linked glycan profile showed fragments from Hex3HexNAc2 to Hex9HexNAc2. Based in our findings and ubiquitin function, we hypothesize that the same ubiquitin Asn-Asp-Ser sequon carries heterogenic glycosylations, at different metabolic pathway stages according to ubiquitin functional requirements. Finally, we propose a set of possible high mannose N-glycan structures that will help to elucidate the ubiquitin biochemical composition and may well represent good targets for anti-amoebic drugs.

Combined-modality therapy for pulmonary alveolar proteinosis in a remote setting: a case report.

BACKGROUND: Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by accumulation of phospholipoproteinaceous material in the alveoli. The presentation is nonspecific but typically includes dyspnea; the spectrum of disease includes rapidly progressive hypoxic respiratory failure. Whole lung lavage (WLL) is the treatment of choice in symptomatic PAP, but transient worsening of oxygenation sometimes requires salvage modalities of support such as extracorporeal membrane oxygenation (ECMO). Granulocyte macrophage colony-stimulating factor (GM-CSF) plays a role in the pathophysiology of PAP. We highlight a case of severe PAP treated with exogenous GM-CSF and sequential lobar lavage due to the unavailability of salvage methods of oxygenation. CASE PRESENTATION: A 36 year old female was admitted with fevers, chills, and progressive dyspnea. On presentation she was tachypneic, tachycardic, and hypoxemic; labs revealed leukocytosis and lactic acidosis. Chest CT identified diffuse ground glass opacities in a 'crazy-paving' pattern. Following intubation due to impending respiratory failure, bronchoscopy with bronchoalveolar lavage was performed. The lavage return stained positive with Periodic Acid Schiff, confirming the diagnosis of PAP. Continued deterioration necessitated treatment; however, at this geographically remote center without ECMO services WLL was judged to carry significant risk. Nebulized GM-CSF was administered without significant improvement. Subcutaneous GM-CSF was administered and isolated subsegmental lavages of the bilateral upper lobes were performed, with rapid improvement in oxygenation. Additional sequential lobar lavage and continued GM-CSF therapy as an outpatient resulted in complete resolution of oxygen requirement and return to normal pulmonary physiology. CONCLUSIONS: The autoimmune form of PAP is the most common, indicating that therapy with GM-CSF may play an important role for many patients. Treatment with WLL may be impractical in some clinical settings due to the expertise and salvage modalities required. Sequential lobar lavage requires less specialized expertise and may incur less risk of refractory hypoxemia. We posit that this combined-modality therapy is ideally suited to geographically-remote centers such as our own.

Methacarn preserves mucus integrity and improves visualization of amoebae in gills of Atlantic salmon (Salmo salar L.).

Two aqueous fixation methods (modified Davidson's solution and modified Davidson's solution with 2% (w/v) Alcian blue) were compared against two non-aqueous fixation methods (methacarn solution and methacarn solution with 2% (w/v) Alcian blue) along with the standard buffered formalin fixation method to (a) improve preservation of the mucous coat on Atlantic salmon, Salmo salar L., gills and (b) to examine the interaction between the amoebae and mucus on the gill during an infection with amoebic gill disease. Aqueous fixatives demonstrated excellent cytological preservation but failed to deliver the preservation of the mucus when compared to the non-aqueous-based fixatives; qualitative and semi-quantitative analysis revealed a greater preservation of the gill mucus using the non-aqueous methacarn solution. A combination of this fixation method and an Alcian blue/Periodic acid-Schiff staining was tested in gills of Atlantic salmon infected with amoebic gill disease; lectin labelling was also used to confirm the mucus preservation in the methacarn-fixed tissue. Amoebae were observed closely associated with the mucus demonstrating that the techniques employed for preservation of the mucous coat can indeed avoid the loss of potential mucus-embedded parasites, thus providing a better understanding of the relationship between the mucus and parasite.

Morphological characterization of the adherence and invasion of Streptococcus agalactiae to the intestinal mucosa of tilapia Oreochromis sp.: An in vitro model.

Streptococcosis in tilapia Oreochromis sp. is possibly the most important bacterial disease for fish production worldwide. In Colombia, streptococcosis is caused by Streptococcus agalactiae (GBS), but in other countries, Streptococcus iniae is also involved. Prevention of streptococcosis is required and must be addressed for economic, social, international trade and public health reasons. This research used an in vitro culture of tilapia intestine to detail the intestinal mucosal response once the pathogen contacts the epithelium. We show that S. agalactiae sheds off its capsule to adhere to the epithelium. The bacterium adheres as a single individuum, in groups or in chains and is able to divide on the apical border of enterocytes. GBS adheres at and invades exclusively through the apical portion of the intestinal folds, using the transepithelial route. Once within the cytoplasm of enterocytes, the bacteria continue to divide. On the basolateral side of the epithelium, the microorganisms leave the cells to reach the propria and travel through the microcirculation. No evidence of an immuno-inflammatory reaction or goblet cell response in the epithelium or the lamina propria was seen during the process of adherence and invasion of the pathogen.

Distinguishing Epidermolysis Bullosa Acquisita From Bullous Pemphigoid Without Direct Immunofluorescence.

BACKGROUND: It has been postulated that periodic acid-Schiff staining of basement membrane can predict direct immunofluorescence patterns seen in epidermolysis bullosa acquisita and bullous pemphigoid. It has also been suggested that the type of inflammatory infiltrate or presence of fraying of basal keratinocytes may differentiate these two conditions. OBJECTIVE: In this study, we aimed to confirm these observations. METHODS: We reviewed 13 cases of direct immunofluorescence-confirmed epidermolysis bullosa acquisita and 19 cases of direct immunofluorescence-confirmed bullous pemphigoid, all with a subepidermal blister in the routinely processed specimen. The gold standard for diagnosis of epidermolysis bullosa acquisita vs bullous pemphigoid was taken to be identification of immune deposits on the dermal side ('floor' for epidermolysis bullosa acquisita) or the epidermal side ('roof' for bullous pemphigoid) of the salt-split direct immunofluorescence specimen. Our tests to distinguish epidermolysis bullosa acquisita from bullous pemphigoid on the routinely processed biopsy included periodic acid-Schiff basement membrane on the blister roof, neutrophilic infiltrate, lack of eosinophilic infiltrate, and absence of keratinocyte fraying. RESULTS: Sensitivity and specificity for each test were as follows: periodic acid-Schiff staining of roof (sensitivity 25%, specificity 95%), neutrophilic infiltrate (sensitivity 54%, specificity 74%), lack of eosinophilic infiltrate (sensitivity 92%, specificity 68%), and absence of keratinocyte fraying (sensitivity 62%, specificity 58%). CONCLUSIONS: Features in the routinely processed biopsy were unable to reliably distinguish between epidermolysis bullosa acquisita and bullous pemphigoid. Direct immunofluorescence on salt-split skin remains the standard for differentiation.

Confirmatory Testing Prior to Initiating Onychomycosis Therapy Is Cost-Effective.

BACKGROUND: Onychomycosis can be investigated by sampling. Information gleaned includes nail bed involvement, nail plate penetration, fungal viability, and species identification. Testing samples can confirm a diagnosis. While diagnostic testing is considered useful in directing therapy, a substantial number of clinicians do not confirm diagnosis prior to treatment. OBJECTIVES: The aim of this study is to quantify the benefit of confirmatory testing prior to treating toenail onychomycosis. METHODS: The cost of mycological cure (negative potassium hydroxide and negative culture) and the cost-effectiveness of confirmatory testing were determined using the average cost of potassium hydroxide (KOH), culture, periodic acid-Schiff (PAS), efinaconazole, ciclopirox, terbinafine, and itraconazole. Costs were obtained through literature searches, public domain websites, and telephone surveys to local pharmacies and laboratories. To represent the potential risks of prescribing onychomycosis treatment, the costs associated with liver monitoring, potential life-threatening adverse events, and drug-drug interactions were obtained through public domain websites, published studies, and product inserts. RESULTS: PAS was determined to be the most sensitive confirmatory test and KOH the least expensive. The overall cost of an incorrect diagnosis (no confirmatory test used) ranged between $350 and $1175 CAD per patient for treatment of 3 infected toenails. Comparatively, performing confirmatory testing prior to treatment decreases the overall cost to $320 to $930, depending on the therapy, physician, and test. CONCLUSIONS: It is preferred to diagnose onychomycosis prior to treatment. Furthermore, there are cost savings when confirmatory testing is performed before initiating treatment with both topical and oral antifungals in Canada.

A novel application of periodic acid-Schiff (PAS) staining and fluorescence imaging for analysing tapetum and microspore development.

The precisely timed process of tapetum development and its degradation involving programmed cell death is an important molecular event during anther development. Through its degeneration, the tapetum not only provides nutritive substances to the developing microspores but also contributes to the pollen wall by way of sporopollenin, which is a complex mixture of biopolymers, containing long-chain fatty acids, phenylpropanoids, phenolics and traces of carotenoids. A number of dyes and staining methods have been used to visualize tapetal structure and its components by using light microscopy techniques, but none of these methods could differentially stain and thus distinguish tapetal cells from other cell types of anther wall. While analysing progression of tapetum development in different cell types in rice anthers, we discovered a unique property of periodic acid-Schiff (PAS) stain, which upon interaction with some specific component(s) in tapetal cells and developing microspores emits fluorescence at ~620 nm. In rice anthers, the PAS-associated fluorescence could be observed initially in tapetum and developing microspores, and subsequent to degeneration of tapetum, the fluorescence was found to emanate mainly from the pollen wall. We also show that PAS-dependent fluorescence in tapetal cells is distinct from the autofluorescence resulting from pollen wall components and is also not caused by interaction of PAS with pollen starch. Henceforth, this novel fluorescence property of PAS stain could prove to be a new tool in the toolkit of developmental biologists to analyse different aspects of tapetum development and its degeneration with little more ease and specificity.