Comparative performance evaluation of four different methods for diagnosing Giardia infection in dogs and zoonotic assemblages' identification.

Giardia duodenalis (syn. G. intestinalis or G. lamblia) is a parasitic protozoan that infects the upper intestinal tract of a broad range of hosts, including humans and domestic animals. Thus, it has raised concerns about the public health risk due to companion animals. Recently, with the improvement of living standards and increasing contacts between pets and humans, the zoonotic transmission of Giardia has dramatically increased. From a genetic point of view, G. duodenalis should be viewed as a complex species that includes eight different species-specific genetic assemblages. The laboratory diagnosis is mainly based on the finding of microscopic cysts in stool samples by coprological examination. Other methods include the detection of antigens, immunoassays or PCR protocols, which allow the identification of Giardia assemblages. The study aimed to compare the performance of Direct Fluorescence Antibody test (DFA), zinc sulfate flotation technique (ZnSO4), rapid diagnostic test (RDT), end-point PCR amplification (PCR) for the detection of Giardia and to identify the concerning assemblages in a canine population from Central Italy. Direct fluorescence antibody test is the reference standard for laboratory diagnosis of Giardia in fecal samples from dogs, despite the microscopic examination after flotation remains the most useful method in many veterinary diagnostic centers. The present findings demonstrate the high performance of DFA and ZnSO4 in detecting Giardia, while RDT may be useful as alternative or complementary method to the DFA and ZnSO4. PCR performance was low, but it allowed determining Giardia B zoonotic assemblage in 25% of the PCR-positive specimens (15 out of 60), while the remaining PCR-positive isolates belonged to the dog-specific assemblage C. The 26% prevalence of G. duodenalis detected by DFA in owned dogs and the identification of potentially zoonotic assemblages underline the potential risk for public health and indicate frequent cross-species transmission of the parasite between humans and dogs.

Ocular Cicatricial Pemphigoid in Young Patients.

PURPOSE: The purpose of this study is to report ocular cicatricial pemphigoid (OCP) occurring in young patients. Relevant literature is also reviewed. METHODS: Medical records of patients aged 30 years or younger who were treated for OCP between August 2021 and May 2023 at a tertiary care eye institute were reviewed. The most common differential diagnoses of cicatrizing conjunctivitis, such as Stevens-Johnson syndrome sequelae, chemical injury, chronic topical/systemic drug use, autoimmune connective tissue disorders, or allergic eye diseases, were ruled out based on clinical history, examination, and the Cicatrizing Conjunctivitis Score described by Shanbhag et al. The diagnosis of OCP was confirmed by positive direct immunofluorescence of oral mucosal and/or conjunctival biopsy in a majority of the patients. RESULTS: Seven patients fulfilled the criteria for a diagnosis of OCP. The mean age at presentation was 21.86 ± 5.25 years (13-28 years). Some of the patients presented with relatively atypical features for OCP such as corneal immune ring infiltrate and bilateral corneal perforation. Six patients exhibited systemic mucosal lesions, and the direct immunofluorescence yield was 85.71%. All patients required aggressive immunosuppressive treatment. CONCLUSIONS: OCP is classically described as a rare disease that occurs in old age. This case series highlights the importance of a higher index of suspicion for diagnosing OCP at a younger age. Early administration of immunosuppressive agents can potentially control severe ocular surface inflammation and its sequelae.

Retrospective study of laboratory-based surveillance of rabies in wild and domestic animals in the southern United States, 2010-2021.

We performed a retrospective study of all case submissions for the rabies virus (RABV) direct fluorescent antibody test (DFAT) requested of the Tifton Veterinary Diagnostic and Investigational Laboratory (Tifton, GA, USA) between July 2010 and June 2021. Submitted were 792 samples from 23 animal species from 89 counties in Georgia, and 4 neighboring counties in Florida, 1 in South Carolina, and 1 in Alabama. In 13 (1.6%) cases, the DFAT result was inconclusive; 779 (98.4%) cases had a conclusive (positive or negative) test result. Of these 779 cases, 79 (10.1%) tested positive across 10 species. The remaining 700 (89.9%) cases were negative. The main reason for submission for RABV testing was human exposure to a potentially rabid animal in 414 (52.3%) cases. Among the 79 positive cases, 74 (93.7%) involved wildlife; raccoons (51 cases; 68.9%) were the primary host confirmed with RABV infection, followed by skunk and fox (8 cases each; 10.8%), bobcat (5 cases; 6.8%), and bats (2 cases; 2.7%). Only 5 domestic animals (6.3% of the positive cases) tested positive during our study period; one from each of the bovine, canine, caprine, equine, and feline species. Hence, the sylvatic cycle plays the predominant role in circulating RABV infection in our study area.

A deep learning approach to direct immunofluorescence pattern recognition in autoimmune bullous diseases.

BACKGROUND: Artificial intelligence (AI) is reshaping healthcare, using machine and deep learning (DL) to enhance disease management. Dermatology has seen improved diagnostics, particularly in skin cancer detection, through the integration of AI. However, the potential of AI in automating immunofluorescence imaging for autoimmune bullous skin diseases (AIBDs) remains untapped. While direct immunofluorescence (DIF) supports diagnosis, its manual interpretation can hinder efficiency. The use of DL to classify DIF patterns automatically, including the intercellular (ICP) and linear pattern (LP), holds promise for improving the diagnosis of AIBDs. OBJECTIVES: To develop AI algorithms for automated classification of AIBD DIF patterns, such as ICP and LP, in order to enhance diagnostic accuracy, streamline disease management and improve patient outcomes through DL-driven immunofluorescence interpretation. METHODS: We collected immunofluorescence images from skin biopsies of patients suspected of having an AIBD between January 2022 and January 2024. Skin tissue was obtained via a 5-mm punch biopsy, prepared for DIF. Experienced dermatologists classified the images as ICP, LP or negative. To evaluate our DL approach, we divided the images into training (n = 436) and test sets (n = 93). We employed transfer learning with pretrained deep neural networks and conducted fivefold cross-validation to assess model performance. Our dataset's class imbalance was addressed using weighted loss and data augmentation strategies. The models were trained for 50 epochs using Pytorch, achieving an image size of 224 × 224 pixels for both convolutional neural networks (CNNs) and the Swin Transformer. RESULTS: Our study compared six CNNs and the Swin Transformer for AIBD image classification, with the Swin Transformer achieving the highest average validation accuracy (98.5%). On a separate test set, the best model attained an accuracy of 94.6%, demonstrating 95.3% sensitivity and 97.5% specificity across AIBD classes. Visualization with Grad-CAM (class activation mapping) highlighted the model's reliance on characteristic patterns for accurate classification. CONCLUSIONS: The study highlighted the accuracy of CNNs in identifying DIF features. This approach aids automated analysis and reporting, offering reproducibility, speed, data handling and cost-efficiency. Integrating DL into skin immunofluorescence promises precise diagnostics and streamlined reporting in this branch of dermatology.

Artificial intelligence (AI) is transforming healthcare through machine and deep learning (computer systems that can learn and adapt, and make complex decisions, without receiving explicit instructions), improving disease management in dermatology, particularly in detecting skin cancer. However, AI's potential in automating immunofluorescence imaging in autoimmune bullous (blistering) skin diseases (AIBDs) remains largely untapped. Manual interpretation of direct immunofluorescence (DIF ­ a type of microscopy) can reduce efficiency. However, using deep learning to automatically classify DIF patterns (for example, the 'intercellular pattern' (ICP) and the 'linear pattern' (LP)) holds promise in helping with the diagnosis of AIBDs. This study aimed to develop AI algorithms for the automated classification of AIBD DIF patterns, such as ICP and LP, to improve diagnostic accuracy and streamline disease management. Immunofluorescence images were collected from skin biopsies of patients with a suspected AIBD between January 2022 and January 2024. Dermatologists classified the images into three categories: ICP, LP and negative. The dataset was divided into training (436 images) and test sets (93 images). A transfer learning framework (where what has been learned previously in one setting is used to improve performance in another) was used to make up for the limited amount of training data, to explore different models for the AIBD classification task. Our results revealed that a model called the 'Swin Transformer' achieved an average accuracy of 99% in diagnosing different AIBDs. The best model attained 95% accuracy on the test set and was reliable in identifying and ruling out different AIBDs. Visualization with Grad-CAM (a technique used in deep learning) highlighted the model's use of characteristic patterns to classify the diseases accurately. Overall, integrating deep learning in skin immunofluorescence promises to improve diagnostics and streamline reporting in dermatology, which could improve consistency, speed and cost-efficiency.

Concordance of clinical, histopathologic and direct Immunofluorescence findings in patients with intraepidermal immunobullous disorders.

Objective: To determine the concordance among clinical, histopathological and immunofluorescence as diagnostic methods for intraepidermal immunobullous disorders. METHODS: The prospective cross-sectional study was conducted at the Institute of Skin Diseases, Karachi, from December 2020 to December 2022, and comprised adult patients of either gender presenting with complaints of bullae, vesicles, pustules and crusts on the skin or mucous membrane. Diagnostic findings of each patient as obtained by clinical assessment, microscopy and direct immunofluorescence were compared. Data was analysed using SPSS 19. RESULTS: Of the 81 patients, 41(50.6%) were males and 40(49.4%) were females. The overall median age was 35 years (interquartile range: 23 years), with 66(75%) patients aged 19-55 years. The predominant body site involved was the trunk 49(60.5%), followed by mucosa 26(32.1%). Clinical diagnosis detected 80(98.7%) cases, compared to 76(93.8%) by microscopy and 81(100%) by direct immunofluorescence. Conclusion: Direct immunofluorescence was found to be the gold standard for a confirmatory diagnosis of intraepidermal immunobullous disorders, especially when clinical and histopathology findings were inconclusive.

Low pH condition impairs BP-IgG binding to the basement membrane zone.

Bullous pemphigoid (BP), an autoimmune subepidermal blistering disease, shows tense blisters associated with urticarial erythema. Tissue-bound Immunoglobulin G (IgG) at the basement membrane zone (BMZ) detected by direct immunofluorescence (DIF) is strong evidence for a diagnosis of BP. The sensitivity of DIF is higher in complement component 3 (C3) than in IgG, but the reason for this different sensitivity is not fully understood. In this study, we performed several ex vivo studies to investigate the possible mechanism of IgG negativity and C3 positivity at the BMZ by DIF in some BP cases. First, sera from BP patients showing IgG negativity by DIF were found to clearly react to the BMZ in their own DIF skin samples. Next, indirect immunofluorescence (IIF) was performed using sera diluted with different pH phosphate-buffered saline (PBS), pH 7.4, 6.0, and 3.0. Patients' sera diluted with pH 7.4 PBS showed linear staining at the BMZ, but sera diluted with pH 6.0 PBS and pH 3.0 PBS showed lower fluorescence intensities. Finally, sections of skin from BP patients were pre-incubated with different pH PBS (pH 3.0, 6.0, and 7.4), followed by staining with anti-human IgG and C3. The fluorescence intensities were notably lower for IgG and C3 that had been pre-incubated with pH 3.0 PBS and pH 6.0 PBS than for IgG and C3 that had been pre-incubated with pH 7.4 PBS. These results suggest that a low pH condition hinders the binding of autoantibodies to the BMZ, that is, the drop in tissue pH induced by inflammation inhibits autoantibodies from depositing at the BMZ. Furthermore, the drop in tissue pH causes tissue-bound autoantibodies to detach from the BMZ. Complement fragments are activated not only on IgG but also on the cell surface of cells close to IgG during complement activation. IgG may detach from the BMZ under low pH condition induced by inflammation, but some complement fragments remain at the BMZ. These phenomena may help to explain why C3 is more sensitive than IgG when DIF is used to diagnose BP.

Direct immunofluorescence (DIF) versus immunohistochemical (IHC) staining of complements and immunoglobulins (Ig) in pemphigus group.

INTRODUCTION: Pemphigus is a group of bullous disorders of the skin characterized by the formation of autoantibodies present in the intercellular junction of the epidermis. Diagnosis is made by clinical, histopathological examination, and DIF. As DIF needs frozen sections, fluorescent tagged antibodies, UV light microscope for examination, and trained personnel, its non-availability makes a definitive diagnosis challenging. AIMS AND OBJECTIVES: To evaluate the utility of IHC staining of complements and Ig in cases of Pemphigus. MATERIALS AND METHODS: Twenty-six diagnosed cases of Pemphigus were stained by Peroxidase immunohistochemical method using monoclonal antibody to IgG, IgA, IgM, IgG4, C3, C4 d with DAB as chromogen. Pemphigus cases include twenty of pemphigus vulgaris (PV), four cases of pemphigus foliaceous (PF), and two of pemphigus vegetans (Pveg). Positivity was defined as the deposition of Ig and complements as distinct, continuous brown staining of keratinocytes at intercellular junctions. RESULT: On IHC total of 20 PV 17 showed positivity (85%) for IgG, 11 (55%) C4d, 19 (95%) C3d, and 16 (80%) IgG4 deposits at the intercellular junction of the epidermis. All cases of PF showed a deposit of IgG, with three (75%) cases for IgG4, C3d, and C4d. Both cases of Pveg showed positivity for IgG and C4d while one case was negative for IgG4 and C3d. The overall IgG, C3, IgG4, and C4d expression for pemphigus was seen in 88%, 88%, 76.9%, and 61.5% of cases. The relation between these markers, combination of IgG and C3, was best related to each other ( P value = 0.80). The sensitivities for IgG, IgG4, and C3 were 77.8%%, 73%, and 73% resp. CONCLUSION: We conclude that IHC is a useful tool in the diagnosis of PV with the highest sensitivity of IgG and C3d. The combination of IgG and C3d could replace the DIF in almost all of our cases, so IHC on FFPE sections be used as an alternative method to DIF.

To interpret and analyze the changing patterns of histology and direct immunofluorescence findings in membranoproliferative glomerulonephritis.

Background: Membranoproliferative glomerulonephritis has in the recent past been regrouped into immune complex-mediated (ICM MPGN) disease (driven by the classical complement pathway) and complement-mediated (C3GN) disease (driven by the alternative complement pathway) based on pathogenetic role of alternative complement pathway and immunofluorescence deposits. The proposed regrouping lent therapeutic and prognostic support in managing the disease of MPGN. Aims and Objectives: The present study is undertaken to study the patterns of MPGN based on histopathological and DIF examination and sub-categorize the cases into mainly complement dominant and immune complex-mediated diseases for better prognostic and therapeutic utility. Materials and Methods: This is a prospective observational study carried out in a tertiary care center over a period of 2 yrs. The clinically suspected cases of MPGN were subjected to histopathologic and direct immunofluorescence examination (DIF), and the findings were interpreted in light of complement-mediated and immune complex-mediated MPGN. Results: Out of 620 renal biopsies, diagnosis of MPGN was confirmed both on histopathology and DIF in 36 cases accounting for 5.8% of all biopsies. Based on DIF findings, the various groups comprised 20 cases (55.6%) of immune complex deposits, 11 (30.5%) of C3 dominant picture, and 5 (13.9%) of Nil immune deposits. On analysis of the patterns on DIF, 16 cases (80%) of C3 + Ig group and 6 (54.5%) of C3GN group showed predominantly MPGN pattern. Crescentic glomerulonephritis, global glomerulosclerosis, and interstitial fibrosis were markedly observed in C3GN group. Conclusion: DIF is of immense prognostic and therapeutic value in managing cases of MPGN.

Direct immunofluorescence on formalin-fixed, paraffin-embedded versus fresh frozen human renal biopsies: a comparative study.

BACKGROUND: The data referring to the value of direct immunofluorescence on formalin-fixed, paraffin-embedded tissue (IF-Paraffin) in the diagnosis of renal diseases is controversial. The aim of this study was to investigate whether renal biopsies evaluated by routine immunofluorescence on frozen tissue (IF-Frozen) would yield adequate findings to confirm diagnoses when the IF-Paraffin technique was applied. METHODS: To show immunoglobulins, complement components, and light chains, 55 native renal biopsies were subjected to IF-Paraffin and IF-Frozen staining techniques. The intensity of the staining was compared, and the sensitivity and specificity were calculated. RESULTS: The IF-Paraffin technique showed a sensitivity of 89%, 81%, 86%, 30%, 71%, 60%, and 77% for IgG, IgM, IgA, C1q, C3, κ, and λ, respectively, whereas specificity was 91%, 100%, 100%, 96%, 94%, 98%, and 100%. It showed diagnostic findings in 87% of cases. Compared to cases that had both IF-Paraffin and IF-Frozen staining techniques, 43 of 55 showed either equal intensity for the diagnostic immunoglobulin/complement or a little difference. CONCLUSIONS: Direct immunofluorescence on formalin-fixed, paraffin-embedded sections cannot replace immunofluorescence on frozen sections in the assessment of renal biopsies, but may be a "salvage technique" when frozen tissue is insufficient or unavailable and must be interpreted with great caution.

Direct Immunofluorescence Findings and Factors Affecting Conjunctival Biopsy Positivity in Ocular Mucous Membrane Pemphigoid.

PURPOSE: The aim of this study was to describe the direct immunofluorescence (DIF) findings and factors affecting conjunctival biopsy positivity in patients clinically diagnosed with ocular mucous membrane pemphigoid (OMMP). METHODS: This retrospective observational case series included patients with clinical OMMP who underwent conjunctival biopsy for DIF in at least 1 eye between 2018 and 2021 in an institutional setting. The primary outcome measures were association of age and chronic ocular complications with biopsy positivity. RESULTS: Of 61 patients, DIF positivity was seen in 33 (54.1%) clinically suspected cases of OMMP. Of 39 patients who underwent bilateral biopsy, 23 (59%) were positive, of which 12 (52%) were positive in both eyes while 11 (48%) were positive in 1 eye. Of 22 patients who underwent unilateral biopsy, 10 (45%) were positive. Of the 100 biopsied eyes, 45 (45%) were DIF positive. Among the immunoreactants studied, linear deposition of C3 was seen in all 45 positive eyes (100%). Increasing age was significantly associated with higher likelihood of biopsy negativity ( P = 0.032), whereas a greater Sotozono chronic ocular complication score, indicative of disease severity, was associated with low likelihood of biopsy positivity ( P = 0.0042) and lower overall expression of immunoreactants on DIF ( P = 0.0007). CONCLUSIONS: Older patients and patients with more severe ocular surface disease sequelae are likely to have negative DIF results. To optimize the chances of confirming the diagnosis of OMMP by DIF, both eyes should be biopsied early in the disease course. If 1 eye is being biopsied, the less affected eye must be chosen.

Survey: Preferred practice patterns in the diagnosis of mucous membrane pemphigoid amongst cornea specialists.

PURPOSE: To evaluate preferred diagnostic tools and treatment decision-making factors in cases suspicious of mucous membrane pemphigoid (MMP) amongst ophthalmologists and cornea specialists. METHODS: Web-based survey, consisting of 14 multiple choice questions, posted to the Cornea Society Listserv Keranet, the Canadian Ophthalmological Society Cornea Listserv, and the Bowman Club Listserv. RESULTS: One hundred and thirty-eight ophthalmologists participated in the survey. Eighty-six percent (86%) of respondents were cornea trained and practiced in either North America or Europe (83%). Most respondents (72%) routinely perform conjunctival biopsies for all suspicious cases of MMP. For those who do not, fear that biopsy will exacerbate inflammation was the most common reason to defer investigation (47%). Seventy-one percent (71%) performed biopsies from perilesional sites. Ninety-seven percent (97%) ask for direct (DIF) studies and 60% for histopathology in formalin. Most do not recommend biopsy at other non-ocular sites (75%), nor do they perform indirect immunofluorescence for serum autoantibodies (68%). Immune-modulatory therapy is started following positive biopsy results for most (66%), albeit most (62%) would not let a negative DIF influence the choice of starting treatment should there be clinical suspicion of MMP. Differences in practice patterns as they relate to level of experience and geographical location are contrasted to the most up-to-date available guidelines. CONCLUSION: Responses to the survey suggest that there is heterogeneity in certain practice patterns for MMP. Biopsy remains an area of controversy in dictating treatment plans. Identified areas of need should be targeted in future research.

Demonstration of direct immunofluorescence on three substrates in a case of pemphigus.

Direct immunofluorescence (DIF) on skin is considered as the gold standard in the diagnosis of pemphigus. However, alternate substrates can be used. We demonstrate DIF on three substrates, skin biopsy specimen, anagen hair and scrapings of oral erosions. Collection of alternative substrates can be more acceptable to young patients as it is less invasive. It may also be used to detect relapses in cases of pemphigus.

A retrospective study on the direct immunofluorescence findings in pigmented purpuric dermatosis.

BACKGROUND: Pigmented purpuric dermatosis (PPD) is characterized by grouped petechiae, purpuric macules, and pigmentation in the bilateral lower extremities. It runs a chronic and relapsing course. Pathophysiology is poorly understood, but it has been proposed to be an immune-complex disease or capillaritis. This study aimed to determine the incidence and patterns of positive direct immunofluorescence (DIF) findings in patients with clinically and histopathologically confirmed PPD. The association between DIF deposition type and clinical profile was also analyzed. METHODS: Patients with a clinical and histopathologic PPD diagnosis who had undergone DIF studies at a tertiary medical center with attached dermatopathology and immunofluorescence diagnostic centers between January 2002 and December 2021 were included in this study. Data on age, sex, disease duration, comorbidities, and drug intake were collected from medical records. RESULTS: There were 65 patients who satisfied the inclusion criteria. Among them, 58 (89%) had at least one positive finding and 53 (82%) were vascular deposition of immunoglobulin (Ig), complement, or fibrinogen. The most common vascular deposition was fibrinogen (71%) followed by C3 (62%), IgM (18%), IgA (6%), and IgG (3%). Fibrinogen deposition was associated with hypertension (p < 0.03). There was no association between vascular DIF deposition of IgG, IgA, and C3, with age, sex, comorbidities, disease duration, and drug history. CONCLUSION: The most common DIF findings in PPD were vascular deposition of fibrinogen and C3, with or without Ig presence. DIF findings supported a vascular origin in PPD but not an immune complex-mediated disease. Hypertension was associated with fibrinogen deposition and may play a role in its pathophysiology.

Unusual direct immunofluorescence (DIF) pattern in pemphigus herpetiformis- A case report.

Pemphigus herpetiformis (PH) is an autoimmune intraepithelial bullous skin disorder. A 61-year-old female presented with history of multiple pruritic erosions, ulcers all over body, and diffuse loss of hair over scalp. Oral and genital mucosas were uninvolved. Subcorneal separation with suprapapillary thinning of epidermis, neutrophilic spongiosis, and elongation of rete ridges were seen on histopathology. Direct immunofluorescence (DIF) revealed IgG deposits in intercellular zone in fish net like pattern and focal linear IgA deposits along basement zone. Indirect immunofluorescence (IIF) revealed antibodies to desmoglein1 (Dsg-1) positive. A final diagnosis of PH was given. The patient responded well to treatment with dapsone and steroids.

The conventional diagnosis challenge: Real-time PCR and nested PCR correlation with the scoring system for individuals at high-risk of Pneumocystis jirovecii pneumonia / Desafío diagnóstico: PCR anidada y en tiempo real frente a un sistema de puntuación en individuos con gran riesgo de neumonía por Pneumocystis jirovecii

Introduction. Pneumocystis jirovecii is an opportunistic fungus that affects mainly people living with HIV (CD4 cell count lower than 200 cells/ml) and other immunosuppressed patients. Since P. jirovecii does not grow on routine mycological media, diagnosis of P. jirovecii pneumonia relies on indirect evidence of its presence in respiratory samples. Objectives. To associate the results of direct immunofluorescence and two molecular methods with a score to predict P. jirovecii pneumonia in patients with AIDS. Materials and methods. A prospective study was conducted with 40 patients. A respiratory sample collected before treatment was subjected to direct immunofluorescence using the Merifluor kit, to nested PCR targeting the mitochondrial large subunit ribosomal RNA, and to the VIASURE real-time PCR kit. Results. These three techniques revealed P. jirovecii in 6, 12, and 15 samples, respectively. All positive samples by direct immunofluorescence were positive by nested PCR, and all positive samples by nested PCR amplified by real-time PCR. There was a statistically significant association between the P. jirovecii pneumonia score and the molecular methods. Two patients were early diagnosed and responded well to treatment. Conclusion. Molecular methods, especially real-time PCR, are recommended for early diagnosis of P. jirovecii pneumonia in AIDS patients.

Introducción. Pneumocystis jirovecii es un hongo oportunista que afecta principalmente a personas con HIV (recuento de CD4 menor de 200 células/ml) y a otros pacientes inmunosuprimidos. Como P. jirovecii no crece en los medios micológicos de rutina, el diagnóstico de neumonía por P. jirovecii se basa en la evidencia presente en muestras respiratorias. Objetivos. Asociar los resultados de la inmunofluorescencia directa y los de dos métodos moleculares con un puntaje para predecir la neumonía causada por P. jirovecii en pacientes con sida. Materiales y métodos. Se realizó un estudio prospectivo de 40 pacientes. Se recolectó una muestra respiratoria antes del inicio de tratamiento y se sometió a una prueba de inmunofluorescencia directa con el kit Merifluor, una PCR anidada para la amplificación de la subunidad larga del ribosoma mitocondrial y una PCR en tiempo real usando el kit VIASURE. Resultados. Estas tres técnicas evidenciaron la presencia de P. jirovecii en 6, 12 y 15 muestras, respectivamente. Todas las muestras positivas por inmunofluorescencia directa fueron positivas en la PCR anidada y todas las muestras positivas en la PCR anidada amplificaron por PCR en tiempo real. Se encontró una asociación estadística entre los valores de la neumonía causada por P. jirovecii y los métodos moleculares. Dos pacientes con diagnóstico temprano respondieron satisfactoriamente al tratamiento. Conclusión. Se recomiendan los métodos moleculares, especialmente la PCR en tiempo real, para el diagnóstico temprano de neumonía causada por P. jirovecii en pacientes con sida.

Microfluidics-Enabled Isolation and Single-Cell Analysis of Circulating Tumor Cells.

Microfluidic platforms enable the enrichment and analysis of circulating tumor cells (CTCs), a potential biomarker for cancer diagnosis, prognosis, and theragnosis. Combined with immunocytochemistry/immunofluorescence (ICC/IF) assays for CTCs, microfluidics-enabled detection presents a unique opportunity to study tumor heterogeneity and predict treatment response, both of which can help cancer drug development. In this chapter, we detail the protocols and methods employed to fabricate and use a microfluidic device for the enrichment, detection, and analysis of single CTCs from the blood samples of sarcoma patients.

[Diagnostics of ocular mucous membrane pemphigoid]. / Diagnostik des okulären Schleimhautpemphigoids.

The diagnosis of ocular mucous membrane pemphigoid (MMP) remains a challenge as the timing and choice of diagnostic methods have a decisive influence on its quality. A systematic approach is required that includes a comprehensive medical history, critical evaluation of the clinical findings and targeted laboratory testing. The diagnosis is complicated by the fact that some patients present purely clinically the symptoms of MMP without fulfilling the required immunohistochemical and laboratory criteria. Basically, the diagnosis of ocular MMP is based on three pillars: 1) medical history and clinical findings, 2) positive immunohistological (direct immunofluorescence) tissue sample and 3) specific serological autoantibodies. As the diagnosis of ocular MMP often implies prolonged systemic immunomodulatory treatment in predominantly older patients, the accurate diagnosis and approach are of critical relevance. The aim of this article is to present the recently updated diagnostic procedure.

Histopathologic features predictive of perivascular deposition of IgA on direct immunofluorescence in cases of leukocytoclastic vasculitis: A retrospective study of 112 specimens.

IgA vasculitis is a small-vessel vasculitis subtype with increased risk of systemic involvement. We aimed to investigate if any light-microscopic features can predict the presence of perivascular granular IgA deposits on direct immunofluorescence (DIF) microscopy. We performed a retrospective search of cutaneous pathology reports from our internal and consultation practice (January 1, 2010-October 5, 2021) with a diagnosis of leukocytoclastic vasculitis and accompanying DIF. A blinded dermatopathologist reviewed standard microscopy slides for predetermined histopathological features. Fifty-six biopsies (48 patients) and 56 biopsies (42 patients) met inclusion criteria for IgA+ and IgA-, respectively. The presence of eosinophils and mid and deep dermal inflammation were statistically more associated with IgA- (41/56 [73.2%] and 31/56 [55.4%], respectively) than IgA+ cases (28/56 [50.0%] and 14/56 [25.0%]; p = 0.049 and 0.006, respectively, chi-squared test). Other microscopic criteria recorded were not significantly different between the two groups (p > 0.05, chi-squared and Fisher's exact tests). In this retrospective study of 112 cases, we found that while the absence of eosinophils and absence of mid- and deep inflammation were correlated with increased likelihood of IgA perivascular deposition on DIF, no other histopathological features on light microscopy tested could reliably predict the presence of IgA perivascular deposition on DIF. Therefore, DIF remains a necessary component for the accurate diagnosis of cutaneous IgA vasculitis.

Detection of antibody-coated Mucor in skin biopsy by direct immunofluorescence.

Cutaneous mucormycosis may be caused by direct inoculation or hematogenous spread of mucormycetes in immunocompromised patients. Skin biopsy is characterized by a deep fungal infection with frequent angioinvasion. The fungal hyphae can usually be identified on H&E stain. We report a case of cutaneous angioinvasive mucormycosis in which the fungi were also visualized on direct immunofluorescence. A 57-year-old patient with relapsed myelodysplastic syndrome status-post allogeneic hematopoietic cell transplant, diabetes mellitus, and graft-versus-host disease presented with painful, palpable, dark-red to violaceous retiform purpuric plaques. Light microscopy of punch biopsy revealed numerous broad, ribbon-like, pauci-septate hyphae in the dermis with angioinvasion, consistent with mucormycosis. Direct immunofluorescence performed on a concurrent biopsy to exclude immune complex vasculitis showed smooth IgG, IgA (weak), IgM (faint), and C3 deposition on the hyphal structures, compatible with antibody-coated fungi. Tissue culture subsequently confirmed Mucor species. Although mucormycosis was readily diagnosable on routine light microscopy in this case, recognition of the unique phenomenon of antibody-coated fungi can be crucial when the invasive fungi are sparse or only present in the direct immunofluorescence specimen.