Direct Microscopy Research Articles

Comparision of pap stained smears by using auto fluorescence method with ziehl-neelsen method for detection of acid fast bacilli (AFB)

The Ziehl-Neelsen staining method though it plays an important role in detecting acid fast bacilli (AFB) by direct microscopy its low sensitivity makes to look for the other alternatives. : This study was undertaken to evaluate the utility of auto fluorescence method on Papanicolaou stain (Pap) in comparison tothe traditional Ziehl-Neelsen stain (ZN) method in detection of AFB from lymph node aspirates. Fine needle aspirations (FNAs) were done in 153 patients with clinical suspicion of tubercular lymphadenitis. Smears from aspirate were processed for routine cytology for Hematoxyllin & Eosin (H&E), Giemsa, Pap, Ziehl-Neelsen (ZN) and Auramine (AR) staining. Pap stained smears were examined for AFB for their autofluorescence under fluorescent microscope using the blue excitation filter. ZN stained smears are examined for AFB under oil immersion of compound microscope. The efficacy of the autofluorescence method by Pap in detection of acid fast bacilli (AFB) over the conventional Ziehl-Neelsen stain was analyzed by taking AR as a standard control for AFB.Tuberculous lymphadenitis was diagnosed in 36 cases out of 153 clinicall suspected cases by cytology, culture and microscopyThe sensitivity of ZN was 57.14% and specificity was 99.15% while the sensitivity of AF was 96.5% and the specificity was 91.8%. : It was a novel method of detection of AFB, cheaper, easily available and less time consuming than other methods. AR also had high sensitivity rates as AF.

Conventional and molecular diagnostics in onychomycosis-part2 : Molecular identification of causative dermatophytes by polymerase chain reaction and sequence analysis of the internal transcribed spacer region of ribosomal DNA

Dermatophyte identification using traditional methods such as optics-based direct fluorescence microscopy and culture is nowadays supplemented by molecular biological methods. The validity of dermatophyte DNA detection with direct uniplex-polymerase chain reaction-enzyme immunoassay (PCR-EIA) in nail samples was proven by sequence analysis of the ribosomal internal transcribed spacer (ITS) region. A total of 108dermatophytes, isolated from patients with onychomycosis, were positive for Trichophyton rubrum (TR) and Trichophyton interdigitale (TI) in culture and/or uniplex-PCR-EIA. Conventional methods for dermatophyte identification were complemented by direct uniplex-PCR-EIA and sequence analysis of the ribosomal ITS region (18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA). Of 108 patients (average age62, median age 73), 56showed cultural growth with 31of them being identified as TR and23 as TI. There was high agreement with the sequence analysis. Surprisingly, the pathogen of asingle nail sample was identified as T.quinckeanum (formerly T.mentagrophytes sensu stricto), arare zoophilic dermatophyte in Germany. Asingle TI strain turned out to be amisidentified T.tonsurans based on the sequence analysis. In all, 34of the 52specimens lacking cultural growth were detected by PCR as TR, and 18specimens could be identified as TI. The results of dermatophyte identification of culture-negative nail samples were also in agreement with the results of sequence analysis. Molecular biological methods are well applicable, and they show high reliability for direct dermatophyte identification in nail samples without prior cultivation. Especially for nail samples without cultural growth, PCR-based dermatophyte identification was highly specific and sensitive.

A Photoactive Supramolecular Complex Targeting PD-L1 Reveals a Weak Correlation between Photoactivation Efficiency and Receptor Expression Levels in Non-Small-Cell Lung Cancer Tumor Models.

Photo-immunotherapy uses antibodies conjugated to photosensitizers to produce nanostructured constructs endowed with targeting properties and photo-inactivation capabilities towards tumor cells. The superficial receptor density on cancer cells is considered a determining factor for the efficacy of the photodynamic treatment. In this work, we propose the use of a photoactive conjugate that consists of the clinical grade PD-L1-binding monoclonal antibody Atezolizumab, covalently linked to either the well-known photosensitizer eosin or the fluorescent probe Alexa647. Using single-molecule localization microscopy (direct stochastic optical reconstruction microscopy, dSTORM), and an anti-PD-L1 monoclonal antibody labelled with Alexa647, we quantified the density of PD-L1 receptors exposed on the cell surface in two human non-small-cell lung cancer lines (H322 and A549) expressing PD-L1 to a different level. We then investigated if this value correlates with the effectiveness of the photodynamic treatment. The photodynamic treatment of H322 and A549 with the photo-immunoconjugate demonstrated its potential for PDT treatments, but the efficacy did not correlate with the PD-L1 expression levels. Our results provide additional evidence that receptor density does not determine a priori the level of photo-induced cell death.

Clinico Mycological Profile of Dermatophyte Infections at A Tertiary Care Hospital from South India

The prevalence of Dermatophytosis is similar in several continents of the Asian country. The varied state of weather is favourable for mycosis that may lead to various clinical symptoms and infection spreads speedily if left untreated with time. The aim of the present study is to determine the clinical and mycological pattern of dermatophyte infections in patients arriving at our dermatology outpatient department and also to correlate the formal clinical diagnosis with KOH positivity and culture positivity. This descriptive observational study was conducted among 300 patients presenting with dermatophyte infections who came to the dermatology outpatient Department of SRM Medical College and research centre, Kattankulathur, Chengalpattu. The clinical specimens were put through direct microscopy by Potassium Hydroxide (KOH) mount and with the culture on Sabouraud’s Dextrose Agar (SDA). The study showed males were affected mostly and most of the participants were students. The common symptoms observed were Itching, Scaling and Discolouration. Commonly, patients had a primary diagnosis of Tinea corporis infection followed by T. cruris. Trichophyton mentagrophytes were the commonly isolated organism followed by Trichophyton rubrum and Trichophyton violaceum. This study indicates the need for personal hygiene and the disadvantage of as only few participants had zoophilic infections of Tinea species. These methods of diagnosing and identification will further aid in better patients management.

Differences among Indian and European pemphigus patients based on demographics, clinical parameters and propensity for relapse: results of a prospective bicontinental cohort study.

Limited studies have explored pemphigus variations among different ethnic groups residing in their respective geographical locations. This bicontinental study aimed to compare clinical and immunological parameters in Indian and European pemphigus patients in complete remission, off therapy, or on minimal therapy. 105 patients (India, n= 75; Bulgaria, n=15; Greece, n=15) with pemphigus vulgaris (PV) or pemphigus foliaceous (PF) in complete remission on minimal therapy (n=64) or complete remission off therapy (n=41) were recruited. Demographic, clinical, and immunological parameters were compared. Indian patients were significantly younger, the maximal disease severity during the preceding active disease phase was significantly lower, and treatment duration until complete remission was significantly shorter, compared to European patients. European patients had significantly higher anti-Dsg3 serum levels and higher IgG positivity rate based on direct immunofluorescence microscopy at baseline. Furthermore, European patients revealed higher CD19, CD19+ CD27+ cell counts, compared with patients from India. Of note, none of the European patients (n=30) relapsed within the study period, in contrast to 29/75 (38.6%) Indian patients. Treatment strategies differed significantly between the two cohorts, with more frequent utilization of rituximab to achieve remission in the Indian cohort, while prednisolone was more widely used for maintaining remission in the European cohort. The observed heterogeneity of pemphigus among patients of different ethnicities in terms of demographics, clinical parameters, and propensity for relapse may be due to genetic background or different treatment strategies.

Clinical and Mycological Characterization of Chronic and Recurrent Dermatophytes using Various Staining and Microscopic Methods

Dermatophytosis is a prevalent infection in tropical and subtropical countries, including India. This study aims to investigate the epidemiology and clinical aspects of chronic and recurrent dermatophytosis, identify the clinical isolates, and assess the effectiveness of different microscopy and staining methods for diagnosis. The objective of the study is to study the epidemiology and clinical aspects of chronic and recurrent dermatophytosis and to identify the clinical isolates. Attempts to improve the diagnostic outcome by implementing different microscopy and staining methods have also been performed. Adult patients with chronic and recurrent cases and positive direct microscopy were included, and clinical details were recorded. The mycological culture was performed. Fifteen isolates were selected randomly and subjected to 0.9% NaCl, Chicago sky blue staining, Calcofluor white staining, and Congo red staining in addition to the standard lactophenol cotton blue (LPCB) preparation. Among the 178 patients in the study, females (56.7%) and patients aged 25-45 (50%) were more frequently affected. Tinea corporis was the most common clinical type (89.32%). Recurrent cases (56.1%) were more prevalent than chronic cases (43.9%). Culture positivity was seen in 60.1%, with Trichophyton mentagrophytes being the most common isolate (86%). Among the staining methods used on 15 selected isolates, Lactophenol Cotton Blue (LPCB) was scored as the most effective, scoring 2.6 out of 3. The study revealed several clinical and epidemiological findings related to dermatophytosis in India, including a high degree of communicability, inadvertent therapies including topical steroid misuse, and a persistent nature requiring an extended duration of therapy. Trichophyton mentagrophytes was the most frequently isolated pathogen. Of the staining methods evaluated, Lactophenol Cotton Blue (LPCB) was found to be the most effective. The findings suggest a need for continued research into effective treatments and diagnostic methods for dermatophytosis in India.

Detection of Aspergillus Galactomannan in Blood for Early Diagnosis of Pulmonary Aspergillosis in HIV Positive Subjects with and without Tuberculosis in Calabar, Nigeria

This study was aimed at determining the prevalence of pulmonary aspergillosis using Aspergillus galactomannan detection in human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/ AIDS) subjects with and without tuberculosis. The study was a prospective cohort study carried out at the University of Calabar Teaching Hospital and Dr. Lawrence Henshaw Memorial Hospital in Calabar. Subjects were HIV-positive patients with and without tuberculosis. Blood and sputum samples were obtained from 215 subjects who consented to the study. A structured questionnaire was administered to assess demograph¬ic data and medical history. Sputum samples were subjected to direct microscopy, culture, and Ziehl-Nel¬seen test. Aspergillus galactomannan assay and CD4 counts were performed on the blood samples. The results of Aspergillus galactomannan-positive subjects were communicated to the attending Physicians for proper patient management. Aspergillus species were recovered from 35(16.3%) sputum samples. Asper¬gillus fumigatus was the most encountered isolate 18(8.4%). The most common presenting symptoms were cough 35(100%) and fatigue 30(85.7%). Out of the 35 subjects with Aspergillus isolates, 21(60.0%) had TB, 9(25.7%) had no TB and 5(14.3%) had co-morbidity of HIV and TB. All the subjects with aspergillosis had CD4 counts less than 200 cellsμl-1 and there was a statistically significant association between pulmonary aspergillosis and CD4 levels (H=7.02; p = 0.03). Galactomannan assay could be used for early diagnosis of invasive aspergillosis and timely management of patients as it is sensitive, cost-effective, and has a turna¬round time of less than 1 hour compared to culture methods.

Candida isolation and identification from pregnant women in Kirkuk city

Abstract Background: One of the most common fungal species that have been discovered as a component of the normal flora in different human body sections is Candida spp. it is the only major opportunistic fungus that exists in both commensal and pathogenic forms. Objective: This study was prepared to find out the prevalence of candidiasis among pregnant women in Kirkuk city in addition to diagnose and identify its important species. Materials and Methods: This study was carried out on 154 patients whom suspected of having candidiasis and attended at (Azadi Teaching, Kirkuk General, and Al-Nasar Maternity) hospitals from December 2021 till April 2022. Specimens were carried out by taking a vaginal swab from all pregnant women. The swabs were first examined directly by using potassium hydroxide (10%) then inoculated onto Sabouraud’s dextrose agar supported with Amikacine antibiotic. The mycological examination has revealed 62.33% positive candidiasis, by direct microscopy examination through the recognition of pseudohyphae or yeasts. Hi Chrom agar medium was used to detect Candidal species identification. Results: Four species have been isolated each with an incidence of 67.7% for Candida albicans, 13.5% for Candida glabrata, 11.5% for Candida tropicalis, 7.3% for Candida krusie. Conclusion: It can be concluded that Candida is very common among pregnant women and being the most predominant species.

Diagnostic accuracy of qPCR and microscopy for cutaneous leishmaniasis in rural Ecuador: A Bayesian latent class analysis.

Clinical and laboratory diagnosis of cutaneous leishmaniasis (CL) is hampered by under-ascertainment of direct microscopy. This study compared the diagnostic accuracy of qPCR on DNA extracted from filter paper to the accuracy of direct smear slide microscopy in participants presenting with a cutaneous lesion suspected of leishmaniasis to 16 rural healthcare centers in the Ecuadorian Amazon and Pacific regions, from January 2019 to June 2021. We used Bayesian latent class analysis to estimate test sensitivity, specificity, likelihood ratios (LR), and predictive values (PV) with their 95% credible intervals (95%CrI). The impact of sociodemographic and clinical characteristics on predictive values was assessed as a secondary objective. Of 320 initially included participants, paired valid test results were available and included in the diagnostic accuracy analysis for 129 from the Amazon and 185 from the Pacific region. We estimated sensitivity of 68% (95%CrI 49% to 82%) and 73% (95%CrI 73% to 83%) for qPCR, and 51% (95%CrI 36% to 66%) and 76% (95%CrI 65% to 86%) for microscopy in the Amazon and Pacific region, respectively. In the Amazon, with an estimated disease prevalence among participants of 73%, negative PV for qPCR was 54% (95%CrI 5% to 77%) and 44% (95%CrI 4% to 65%) for microscopy. In the Pacific, (prevalence 88%) the negative PV was 34% (95%CrI 3% to 58%) and 37% (95%CrI 3% to 63%). The addition of qPCR parallel to microscopy in the Amazon increases the observed prevalence from 38% to 64% (+26 (95%CrI 19 to 34) percentage points). The accuracy of either qPCR on DNA extracted from filter paper or microscopy for CL diagnosis as a stand-alone test seems to be unsatisfactory and region-dependent. We recommend further studies to confirm the clinically relevant increment found in the diagnostic yield due to the addition of qPCR.

Differential CD38 Expression on Daratumumab-Naïve Vs. Resistant Multiple Myeloma - a Direct Stochastic Optical Reconstruction Microscopy ( dSTORM) Analysis in 53 Patients

Background: The anti-CD38 antibody Daratumumab (Dara) has become the new standard of care for the treatment of newly-diagnosed and relapsed/ refractory multiple myeloma (MM). At the same time, Dara resistance remains poorly understood. Potential resistance mechanisms have been proposed and range from intrinsic downregulation in CD38 dim MM cells to genetic alterations and splicing variants which may affect CD38 epitope expression (Adamia, ASH 2022). Here, we used super-resolution microscopy for high-precision quantification of CD38 in MM and piloted this approach in a large cohort of 53 MM patients with vs. without Dara resistance. Methods: Direct stochastic optical reconstruction microscopy ( dSTORM) is a novel imaging modality based on photoswitching of certain fluorophores coupled to antibodies for molecular representation of surface molecules at unprecedented levels of resolution (<20 nM). To correlate CD38 expression data with clinical outcome, we divided our cohort based on treatment status into two groups: Dara-naïve (n = 31) vs. Dara-resistant (n = 22) patients. Imaging data was complemented by phenotypic assessment using a well-established multi-epitope anti-CD38 antibody (Cytognos), as per standard-of-care. Results: Mean CD38 receptor density as measured with Dara via dSTORM was significantly higher in Dara-naïve (of 48.81 ± 29.65 receptors/µm²) vs. Dara-resistant patients (15.52 ± 3.9 receptors/µm², p = 0.016). This finding was corroborated by multi-epitope phenotypic analysis which revealed mean CD38 receptor expression levels of 32.09 ± 13.14 receptors/µm² in Dara-naïve vs. 21.65 ± 17.73 receptors/µm² in Dara-resistant patients ( p = 0.05). Lower CD38 expression via dSTORM vs. multi-epitope anti-CD38 staining was consistently observed in our cohort and may reflect ongoing blockage of the receptor binding site by Dara in cases with shorter time to last Dara application (median 53 days, range 1-189 days). As there was no correlation with the interval between Dara application and sampling however, splice-site mutations affecting CD38 receptor accessibility as proposed by Adamia et al. may depict a more relevant mechanism of resistance in our Dara-resistant cohort. In a next step, we therefore investigated alternative epitope binding in Dara-resistant patients. To this end, we investigated antibody binding by dSTORM imaging for isatuximab (Isa), an alternative CD38-directed monoclonal antibody that binds to a location in the C-terminal portion of the CD38 extracellular domain which is entirely different from the Dara epitope. Interestingly, there was a trend for increased detection rate with Isa for mean CD38 receptor expression in Dara-resistant patients (22.2 ± 4.6 receptors/µm²) vs. Dara (15.5 ± 3.9 receptors/µm², p = 0.1) and on par with CD38 receptor expression. No data was available to assess Dara binding in Isa pre-exposed patients. Conclusion: Innovative microscopy using dSTORM allows for high-resolution epitope quantification at unprecedented granularity. While this finding enables visual proof of differential epitope binding of Dara vs. Isa on CD38 in a clinically feasible setting, correlation studies to determine the clinical relevance of this observation remain ongoing.

Secondary Genetic Events Impact the Expression of Key Immunotargets on the Surface of Multiple Myeloma Cells

Background: Multiple myeloma (MM) is characterized by complex genomic aberrations including primary translocations of the immunoglobin heavy chain (IGH) locus and secondary mutations involving key oncogenic drivers. This temporal complexity represents a constant challenge for the clinical management of MM, but at the same time may provide new avenues for immunotherapy-based approaches including CAR-T cells and T-cell engaging antibodies. In this study, we investigated the impact of secondary genetic events on the surface expression of common targets for immunotherapy in MM. Methods: Cell models harboring two hotspot activating mutations in KRAS (G12A, A146T) and TP53 (R282W) were created using the MM cell lines OPM-2 and AMO1 via CRISPR-Cas9 technology. Direct stochastic optical reconstruction microscopy ( dSTORM) was used for high-resolution receptor density estimation and epitope quantification of common immunotherapeutic targets such as CD38, BCMA and SLAMF7. Results: dSTORM imaging of cell line models carrying patient-derived secondary genetic alterations revealed a distinct pattern linked to each mutation respectively. In KRAS G12A, we observed a reduction in BCMA density by 2-fold as compared to wild type cells (5.9 ± 0.3 vs. 10.8 ± 1.2 localization clusters/µm 2, p=0005). This reduction in BCMA surface expression was accompanied by a decrease in other key immunotargets, including SLAMF7 (6.0 ± 0.8 vs 11.8 ± 0.8 localization clusters/µm 2, p <0,0001) and CD38 (12.2 ± 1.5 vs 17.9 ± 1.6 localization clusters/µm 2, p=0,012). In KRAS A146, CD38 density increased, whereas mean BCMA and SLAMF7 localizations were consistently reduced as compared to wild type control. To ensure that BCMA downregulation is a consistent feature in genomic high-risk disease, we next simulated a TP53 double-hit situation by introducing an inactivating hotspot mutation R282W in a TP53 deletion background. Surfaceome profiling by dSTORM revealed a minor increase in CD38 levels for TP53R282W/Del as compared to wild type control (51.5 ± 5.7 vs. 36.4 ± 4.2 localization clusters/µm 2, p=0.06). Interestingly, the expression level of BCMA, known to be comparably low on WT tumor cells, further decreased in TP53 double-hit cells (0.6 ± 0.1 vs 1.1 ± 0.1 localization clusters/µm 2, p=0.002), whereas no major differences in the cluster density of SLAMF7 were observed. This leaves room for speculation whether these effects on biallelic TP53 inactivated tumor cells may underlie reduced susceptibility towards BCMA-targeting therapy. Of note, we also observed altered cell morphology in the TP53 double-hit model, characterized by elongated stress fibers enriched with CD138, potentially interfering with receptor accessibility of surface receptors (Figure 1). Recently, secondary genetic events have been reported in BCMA and GPRC5D following targeted immunotherapy inducing acquired drug resistance. We created cell models representing homo- and heterozygous genetic alterations in BCMA and GPRC5D, studies to assess their effects on the surfaceome are ongoing and will be presented at the meeting. Summary: Our data suggest that surface epitope expression in MM cells seems to be less agnostic to secondary genomic events than expected. These findings may provide a direct link between genetic high-risk disease and inferior efficacy of anti-BCMA immunotherapies in the context of e.g. TP53 double-hit MM.

Features of the distribution of hydrological, chemical and biological characteristics in the waters of the Central-East Atlantic in the autumn of 2022 (cruise 63 of the R/V «Akademik Ioffe»)

The goal of the work: to determine the water structure in the Central part of East Atlantic and to identify the features of the distribution of hydrological, chemical and biological characteristics of the upper layer in the autumn periodMaterials and methods: the data presented in the work were obtained on the 63rd cruise of the R/V «Akademik Ioffe» (September 29, 2022–December 8, 2022). During the expedition, hydrological and chemical (temperature, salinity, pH, dissolved oxygen, phosphates, nitrites, nitrates and silicates) characteristics were measured from the surface to the bottom, as well as biological (number, biomass and species diversity of phytoplankton), taken with chemical samples on depths of 0, 10, 20 and 50 m. Chemical analysis was carried out in the ship’s laboratory, according to certified methods for sea and fresh waters. Phytoplankton samples were thickenedusing a reverse filtration funnel through track (nuclear) membranes and fixed with Lugol’s solution to a final concentration of 5%. To study the species diversity, abundance and biomass of plankton microalgae, samples were processed by direct microscopy of algae in a living and thickened drop (V = 0.01 ml), as well as in a counting chamber (V = 0.5 ml) using an Olympus CX43 light microscope (magnification 10×20, 10×40). Identification of taxonomic groups and species composition of microalgae, calculations of the abundance and biomass of phytoplankton, as well as the Sörensen-Czekanowski floristic similarity coefficient according to the adoption of the methods of the « IBSS RAS» were carried out.The practical significance of the research: assessment of the production stock in a poorly studied region of the Central Eastern Caucasus, identification of mechanisms for influencing production processes, assessment of the variability of the water structure against the background of climate change.

First field study using Strong-LAMP for diagnosis of strongyloidiasis in Cubal, Angola

BackgroundStrongyloides stercoralis infection is a common neglected tropical disease distributed worldwide, mainly in tropical and subtropical climates. The impact of S. stercoralis infections on human health ranges from mild asymptomatic infections to chronic strongyloidiasis unnoticeable until the host is immunosuppressed. In severe strongyloidiasis, a syndrome of hyperinfection and larval dissemination to various organs can occur with high mortality rates. The diagnosis of strongyloidiasis is challenging because of the absence of a single standard reference test with high sensitivity and specificity, which also makes it difficult to estimate the accuracy of other diagnostic tests. This study aimed to evaluate, for the first time, the use of an easy-to-perform loop-mediated isothermal amplification (LAMP) colorimetric assay (named Strong-LAMP) for the molecular screening of strongyloidiasis in stool samples from patients in a low-resource endemic area in Cubal, Angola. To compare different LAMP application scenarios, the performance of the Strong-LAMP under field conditions in Angola was reassessed in a well-equipped reference laboratory in Spain and compared with a quantitative polymerase chain reaction (qPCR) method.MethodsA total of 192 stool samples were collected from adult population in Cubal, Angola, and examined by parasitological methods (direct saline microscopy and Baermann’s technique). DNA was extracted from each stool sample using a commercial kit and tested by the colorimetric Strong-LAMP assay for the detection of Strongyloides spp. under field conditions. Furthermore, all samples were shipped to a well-equipped laboratory in Spain, reanalysed by the same procedure and compared with a qPCR method. The overall results after testing were compared.ResultsStrongyloides stercoralis larvae were identified by direct saline microscopy and Baermann in a total of 10/192 (5.2%) and 18/192 (9.4%) stool samples, respectively. Other helminth and protozoan species were also identified. The Strong-LAMP-positive results were visually detected in 69/192 (35.9%) stool samples. The comparison of Strong-LAMP results in field conditions and at a reference laboratory matched in a total of 146/192 (76.0%) samples. A total of 24/192 (12.5%) stool samples tested positive by qPCR.ConclusionsThis is the first study in which colorimetric Strong-LAMP has been clinically evaluated in a resource-poor strongyloidiasis endemic area. Strong-LAMP has been shown to be more effective in screening for strongyloidiasis than parasitological methods under field conditions and qPCR in the laboratory. Our Strong-LAMP has proven to be a field-friendly and highly accurate molecular test for the diagnosis of strongyloidiasis.Graphical

Quantitatively Lighting up the Spatial Organization of CD47/SIRPα Immune Checkpoints on the Cellular Membrane with Single-Molecule Localization Microscopy.

Immunotherapy including immune checkpoint inhibition has reinvigorated the current cancer treatment field. The development of efficient cancer immunotherapies depends on a thorough understanding of the status of immune checkpoints and how they interact. However, the distribution and spatial organization changes of immune checkpoints during their interactions at the single-molecule level remain difficult to directly visualize due to the lack of in situ imaging techniques with appropriate spatial and stoichiometric resolution. Herein, we report the direct visualization and quantification of the spatial distribution and organization of CD47 on the bladder tumor cell membrane and SIRPα on the macrophage membrane by using a single-molecule localization imaging technique called quantitative direct stochastic optical reconstruction microscopy (QdSTORM). Results showed that a portion of CD47 and SIRPα was present on cell membranes as heterogeneous clusters of varying sizes and densities prior to activation. Quantitative analyses of the reconstructed super-resolution images and theoretical simulation revealed that CD47 and SIRPα were reorganized into larger clusters upon binding to each other. Furthermore, we found that blocking the immune checkpoint interaction with small-molecule inhibitors or antibodies significantly impacted the spatial clustering behavior of CD47 on bladder tumor cells, demonstrating the promise of our QdSTORM strategy in elucidating the molecular mechanisms underlying immunotherapy. This work offers a promising strategy to advance our understanding of immune checkpoint state and interactions while also contributing to the fields including signal regulation and cancer therapy.

Prevalence and Predictors Associated to Schistosoma mansoni Infection among Patients Attending the Saint Jean de Malte Hospital, Njombe, Littoral Region, Cameroon.

Schistosomiasis remains a major public health concern in sub-Saharan Africa. It has been associated to morbidity and mortality in developing countries including Cameroon, and Njombe-Penja health district is an endemic area. This study is aimed at determining the prevalence and risk factors of Schistosoma mansoni infection among patients attending the Saint Jean de Malte Hospital, Njombe. A cross-sectional study design was employed, with the enrolment of 300 participants using convenience sampling technique. Data were collected using a structured questionnaire. Stool specimens were collected and examined using direct microscopy, Kato-Katz's method, and formol ether concentration technique. Data were analyzed using SPSS, and chi-square test was used to assess the association. Risk factors for S. mansoni infection were assessed using multivariable logistic regression, and a p < 0.05 was considered significant. The overall prevalence of Schistosoma mansoni infection was 13%. Schistosoma mansoni infection was mostly frequent among patients < 20 years and males. Stream usage (AOR = 2.15, 95% CI. 1.32-3.50), always visiting the stream (AOR = 11.35, 95% CI 2.33-55.33), always swimming and washing clothes in the stream (AOR = 7 : 10, 95% CI 2.31-21.80), age group < 20 years (AOR = 3.7, 95% CI 1.1-12.2), and age group 20-29 years (AOR = 2.58, 95% CI 1.14-18.42) were significantly associated with increased risk of S. mansoni infection. These findings suggest that Schistosoma mansoni infection is of public health concern in Njombe and its environs. Age of <20 years and between 20 and 29 years, stream usage, always visiting the stream, and always swimming and washing clothes in the stream were the main risk factors of S. mansoni infection. Thus, mass drug administration and health education are required.

The Utility of Galactomannan and Polymerase Chain Reaction Assays in Bronchoalveolar Lavage for Diagnosis of Chronic Pulmonary Aspergillosis.

The diagnosis of chronic pulmonary aspergillosis (CPA) is established by combined clinic-radio-microbiological criteria. Out of the different microbiological criteria, a positive serology for Aspergillus-specific IgG levels is the cornerstone of diagnosis. Alternatively, other microbiological evidence are sometimes sought viz., positive Aspergillus antigen (broncho-alveolar lavage fluid,i.e., BALF galactomannan ≥ 1.0), histopathological demonstration of the fungi following lung biopsy or resection, demonstration of hyaline septate hyphae in direct microscopy resembling Aspergillus spp. or its growth on arespiratory specimen. However, the exactroles of BALF- GM and the newer BALF-PCR have not been confirmed by studies till date. This study enrolled 210 patients with suspected CPA. Of the participants, 88 patients met the criteria for CPA, whereas 122 patients had an alternative diagnosis. The sensitivity-specificity of AsperGenius® PCR and "in-house" PCR were 52.27(36.69-67.54) %-33.78 (23.19-45.72) % and 36.36 (22.41-52.23) %-39.19 (28.04-51.23) % respectively. The sensitivity/specificity of BALF (> 1.0) and serum galactomannan (> 1.0) were 46.55% (33.34-60.13)/64.08% (54.03-73.3) and 29.82% (22.05-37.6)/86.84% (81.1-92.59) respectively. The optimal cut-off values for BALF-Galactomannan and serum galactomannan in diagnosing CPA were found to be 0.69 (sensitivity: 64%; specificity: 53%) and 0.458 (sensitivity: 67%; specificity: 64%) respectively. This results of this study suggeststhatAspergillus PCR from BAL may not be a good "rule-in" test for diagnosing CPA. While the performances of GM in BAL and serum may be better than PCR, it should be best used in conjunction with other clinical, radiological, and other microbiological characteristics.

Management of bullous pemphigoid and mucous membrane pemphigoid

Bullous pemphigoid (BP) is the most common blistering autoimmune dermatosis and, as an age-associated disease, is also the bullous dermatosis with the highest increase in incidence in recent years due to demographic developments. Mucous membrane pemphigoid (MMP), which is less common, is aclinically and immunopathologically heterogeneous blistering autoimmune dermatosis characterized by blisters and erosions on mucous membranes. This work summarizes the manifold clinical characteristics of both diseases and provides an up-to-date overview of diagnostics and therapy of BP and SHP. Aselective literature search was carried out. While eczematous, urticarial, or bullous lesions on the integument are typical for BP, MMP is characterized by pronounced, erosive mucosal changes orally, ocularly, genitoanally, tracheally or esophageally and, at most, only minor skin involvement. For diagnosis, the combination of histology, direct immunofluorescence microscopy, and serological detection of autoantibodies against the hemidesmosomal proteins BP180 and BP230 and, in MMP, also against laminin 332 is important. New pathophysiological findings and therapeutics have recently significantly expanded the range of treatment options. Due to the comorbidities of the usually elderly BP patients and the often multiple affected mucous membranes in MMP, with the risk of developing irreversible complications such as strictures and synechiae, interdisciplinary diagnosis and therapy is often required.

Co-infection with Neisseria mucosa in a patient with tuberculous otitis media

Tuberculous otitis media (TOM) is a rare manifestation caused by Mycobacterium tuberculosis with low incidence rates among extrapulmonary tuberculosis cases. Diagnosis is often delayed because of the presence of several clinical manifestations and the high prevalence of secondary bacterial infections. Few reports have attributed secondary bacterial infections in patients with TOM to commensal Neisseria. Thus, understanding the pathogenic mechanisms and clinical features of commensal Neisseria is important, considering its recent presentation as an infection-causing pathogen. Neisseria mucosa is a commensal inhabitant in humans and is generally considered non-pathogenic but can cause infection in rare cases. Here, we report an atypical secondary infection caused by Neisseria mucosa in an 81-year-old woman with TOM being treated for pulmonary tuberculosis. Direct purulent otorrhea smear microscopy revealed no acid-fast bacilli using Ziehl-Neelsen staining, whereas the phagocytosis of gram-negative cocci by white blood cells was confirmed using Gram staining. Otorrhea culture revealed the growth of N. mucosa. Subsequently, M. tuberculosis infection in the otorrhea was identified using a culture-based method. Vigilance is critical for the early detection of TOM to prevent further complications. This report raises awareness regarding TOM and provides insight into the pathogenicity of N. mucosa in otitis media.

Prevalence of Blastocystis and Dientamoeba fragilis in diarrheal patients in Corum, Türkiye.

To investigate the prevalence of Blastocystis and Dientamoeba fragilis in diarrhea patients and healthy individuals in Corum, Türkiye, fecal samples from 92 diarrhea patients and 50 healthy individuals were collected and evaluated using direct microscopy and molecular methods to screen for bacteria, protozoa, and viruses. The prevalence of Blastocystis was 24.6% in total and more frequent in the healthy group (30.0%). The commonly detected STs (subtypes) were ST3 (40.0%) and ST2 (34.2%). The distribution of Blastocystis STs in the healthy and diarrheal groups did not show any difference in sex and age, but ST3 was detected more frequently in patients aged from 40 to 59 years (p < 0.05). Alleles 4 (8/12) and 2 (4/12) were present in ST1; 9 (3/5) and 12 (2/5) in ST2; 34 (9/14), 36 (3/14), and 38 (2/14) in ST3; and only allele 42 (2/2) in ST4. D. fragilis was present in 8.4% of the population. However, there was no statistically significant difference between the healthy and diarrheic groups (12.0% and 6.5%, respectively), neither with respect to age nor sex. Co-infection was 58.3% and was more frequent in healthy individuals (33.3%) than in diarrhea patients (25.0%). Blastocystis ST3 was the most common subtype detected, with D. fragilis at 33.3%. Salmonella, Shigella, or helminth eggs were not observed in all groups, but Entamoeba histolytica, Giardia intestinalis, Cryptosporidium, Rotavirus, Adenovirus, and Clostridium difficile toxin were found only in diarrhea patients. These findings support the hypothesis that Blastocystis and D. fragilis may be part of the healthy human gut microbiome.

Etiological Bacterial Spectrum of Purulent Inflammation of Cervical Lymph Nodes in Children With Unknown Causes of Lymphadenopathy.

In the management of the non-specific purulent inflammation of the cervical lymph nodes in children, not only surgical treatment to evacuate the available pusbut also antibacterial therapy is mandatory. Knowledge of the spectrum of the bacterial causative agents of this disease is of fundamental importance for it. This retrospective study included 66 patients with purulent inflammation of the cervical lymph nodes with a mean age of 5.79 years, ranging from 29 days to 17 years, who were hospitalized for the period 2015-2022 in the three pediatric clinics at the St. Marina University Multispecialty Hospital for Active Treatment in Varna, Bulgaria, and operated by an oral or maxillofacial surgeon, in which for microbiological examination material from the suppurated lymph node was taken and analyzed first by direct microscopy for gram-positivity and then on a biochemical machine identifier VITEK(bioMérieux, Marcy-l'Étoile, France). In all of them, the patients and their parents did not provide anamnestic data explaining the etiology of their infectious diseases of the cervical lymph nodes. There was no clear cause that would explain the occurrence of cervical lymphadenopathy - skin infections, dental diseases, diseases of the ears and upper and lower respiratory tracts, onco-hematological diseases, and others. During the medical examination by pediatricians and surgeons, no entrances to the infection were found, but only local signs of inflammation (redness, swelling, and pain), increased levels of blood markers of inflammation (leukocytes, neutrophils, erythrocyte sedimentation rate, and C-reactive protein) and there was imaging evidence (ultrasound, magnetic resonance imaging, and computed tomography) of a purulent collection in the lymph nodes and/or in the soft tissues surrounding them. Exudate without microbial growth was found in 22 of the cases. The main causative agents of purulent lymphadenopathy are gram-positive bacteria (n=32, 72,73%) - Staphylococcus aureus (n=16), Staphylococcus haemolyticus (n=8), Beta-hemolytic streptococcus(n=2), and gram-positive mixed resident microflora (n=6). However, in 27.27% (n=12) of all 44 patients described in this article with isolated pathogens, cultures were gram-negative. These are Bartonella henselae (n=4), Klebsiella pneumoniae (n=4), Klebsiella oxytoca (n=2), and Flavimonas oryzihabitans (n=2).