Two promising Bacillus-derived antifungal lipopeptide leads AF4 and AF5 and their combined effect with fluconazole on the in vitro Candida glabrata biofilms.

Introduction: Candida species are endowed with the ability to produce biofilms, which is one of the causes of pathogenicity, as biofilms protect yeasts from antifungal drugs. Candida glabrata (Nakaseomyces glabrata) is one of the most prevalent pathogenic yeasts in humans and a biofilm producer. Methods: The study was aimed at evaluating the combined effects of two highly promising antifungal biomolecules (AF4 and AF5) lipopeptide in nature, chromatographically purified to homogeneity from Bacillus subtilis (B. subtilis) and the standard antifungal fluconazole (at different concentrations) to demonstrate C. glabrata biofilm formation inhibition. Biofilm production and inhibition were evaluated by quantification of the biofilm biomass and metabolic activity using crystal violet (CV) staining and XTT reduction assays, respectively. Microscopic techniques such as confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM) were employed to visualize biofilm formation and inhibition. Results and Discussion: Compared to untreated and fluconazole-treated biofilms, an enhanced in vitro anti-biofilm effect of the antifungal lipopeptides AF4/AF5 alone and their combinations with fluconazole was established. The lipopeptides AF4/AF5 alone at 8 and 16 µg/mL exhibited significant biomass and metabolic activity reductions. SEM and CSLM images provided evidence that the lipopeptide exposure results in architectural alterations and a significant reduction of C. glabrata biofilms, whereas (2', 7'-dichlorofluorescin diacetate (DCFDA) and propidium iodide (PI) analyses showed reactive oxygen species (ROS) generation along with membrane permeabilization. The estimation of exopolysaccharides (EPS) in AF4/AF5-treated biofilms indicated EPS reduction. The combinations of fluconazole (64/128 µg/mL) and AF4/AF5 lipopeptide (16 µg/mL) were found to significantly disrupt the mature (24 h) biofilms as revealed by CSLM and SEM studies. The CSLM images of biofilms were validated using COMSTAT. The FTIR-analyses indicate the antibiofilm effects of both lipopeptides on 24 h biofilms to support CSLM and SEM observations. The combinations of fluconazole (64/128 µg/mL) and AF4/AF5 lipopeptide were found to disrupt the mature biofilms; the study also showed that the lipopeptides alone have the potentials to combat C. glabrata biofilms. Taken together, it may be suggested that these lipopeptide leads can be optimized to potentially apply on various surfaces to either reduce or nearly eradicate yeast biofilms.

A novel indirect ELISA for serodiagnosis of mucormycosis using antigens from Rhizopus arrhizus.

BACKGROUND: Due to a delay in diagnosis by conventional techniques and high mortality, the development of a standardised and rapid non-culture-based technique is an unmet need in pulmonary, gastrointestinal, and disseminated forms of mucormycosis. Though limited studies have been conducted for molecular diagnosis, there are no established serologic tests for this highly fatal infection. OBJECTIVE: To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis. METHODS: We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 µg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample. RESULTS: Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity. CONCLUSION: The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.

Innate and adaptive immune responses in subjects with CPA secondary to post-pulmonary tuberculosis lung abnormalities.

BACKGROUND: Post-tuberculosis lung abnormality (PTLA) is the most common risk factor for chronic pulmonary aspergillosis (CPA), and 14%-25% of the subjects with PTLA develop CPA. The pathogenesis and the host immune response in subjects with PTLA who develop CPA need to be better understood. METHODS: We prospectively compared the innate and adaptive immune responses mounted by patients of PTLA with or without CPA (controls). We studied the neutrophil oxidative burst (by dihydrorhodamine 123 test), classic (serum C3 and C4 levels) and alternative (mannose-binding lectin [MBL] protein levels) complement pathway, serum immunoglobulins (IgG, IgM and IgA), B and T lymphocytes and their subsets in subjects with PTLA with or without CPA. RESULTS: We included 111 subjects (58 CPA and 53 controls) in the current study. The mean ± SD age of the study population was 42.6 ± 15.7 years. The cases and controls were matched for age, gender distribution and body weight. Subjects with CPA had impaired neutrophil oxidative burst, lower memory T lymphocytes and impaired Th-1 immune response (lower Th-1 lymphocytes) than controls. We found no significant difference between the two groups in the serum complement levels, MBL levels, B-cell subsets and other T lymphocyte subsets. CONCLUSION: Subjects with CPA secondary to PTLA have impaired neutrophil oxidative burst and a lower Th-1 response than controls.

Carbon substrates promotes stress resistance and drug tolerance in clinical isolates of Candida tropicalis.

Candida tropicalis is a human pathogen and one of the most prevalent non-Candida albicans Candida (NCAC) species causing invasive infections. Azole antifungal resistance in C. tropicalis is also gradually increasing with the increasing incidence of infections. The pathogenic success of C. tropicalis depends on its effective response in the host microenvironment. To become a successful pathogen, cellular metabolism, and physiological status determine the ability of the pathogen to counter diverse stresses inside the host. However, to date, limited knowledge is available on the impact of carbon substrate metabolism on stress adaptation and azole resistance in C. tropicalis. In this study, we determined the impact of glucose, fructose, and sucrose as the sole carbon source on the fluconazole resistance and osmotic (NaCl), oxidative (H2O2) stress adaptation in C. tropicalis clinical isolates. We confirmed that the abundance of carbon substrates influences or increases drug resistance and osmotic and oxidative stress tolerance in C. tropicalis. Additionally, both azole-resistant and susceptible isolates showed similar stress adaptation phenotypes, confirming the equal efficiency of becoming successful pathogens irrespective of drug susceptibility profile. To the best of our knowledge, our study is the first on C. tropicalis to demonstrate the direct relation between carbon substrate metabolism and stress tolerance or drug resistance.

Two promising natural lipopeptides from Bacillus subtilis effectively induced membrane permeabilization in Candida glabrata.

Candida glabrata is an important opportunistic human pathogen well known to develop resistance to antifungal drugs. Due to their numerous desirable qualities, antimicrobial lipopeptides have gained significant attention as promising candidates for antifungal drugs. In the present study, two bioactive lipopeptides (AF4 and AF5 m/z 1071.5 and 1085.5, respectively), coproduced and purified from Bacillus subtilis RLID12.1, consist of seven amino acid residues with lipid moieties. In our previous studies, the reversed phased-HPLC purified lipopeptides demonstrated broad-spectrum of antifungal activities against over 110 Candida albicans, Candida non-albicans and mycelial fungi. Two lipopeptides triggered membrane permeabilization of C. glabrata cells, as confirmed by propidium iodide-based flow cytometry, with PI uptake up to 99% demonstrating fungicidal effects. Metabolic inactivation in treated cells was confirmed by FUN-1-based confocal microscopy. Together, the results indicate that these lipopeptides have potentials to be developed into a new set of antifungals for combating fungal infections.

Impact of sphingolipid synthesis inhibition on the drug susceptibility patterns of Trichophyton species.

The well known dermatophyte infections caused by Trichophyton species are an ambiguous problem to treat using the present arsenal of antifungals. This study expounds on the effect of inhibition of sphingolipid pathway on Trichophyton growth. Findings from the drug susceptibility assays suggest sphingolipid inhibition severely restricts the growth of T. interdigitale and T. tonsurans. The observed synergistic effects of combinations of sphingolipid inhibitor and conventional drugs provide a promising treatment strategy against Trichophyton infection.

Sphingolipid diversity in Candida auris: unraveling interclade and drug resistance fingerprints.

In this study, we explored the sphingolipid (SL) landscape in Candida auris, which plays pivotal roles in fungal biology and drug susceptibility. The composition of SLs exhibited substantial variations at both the SL class and molecular species levels among clade isolates. Utilizing principal component analysis, we successfully differentiated the five clades based on their SL class composition. While phytoceramide (PCer) was uniformly the most abundant SL class in all the isolates, other classes showed significant variations. These variations were not limited to SL class level only as the proportion of different molecular species containing variable number of carbons in fatty acid chains also differed between the isolates. Also a comparative analysis revealed abundance of ceramides and glucosylceramides in fluconazole susceptible isolates. Furthermore, by comparing drug-resistant and susceptible isolates within clade IV, we uncovered significant intraclade differences in key SL classes such as high PCer and low long chain base (LCB) content in resistant strains, underscoring the impact of SL heterogeneity on drug resistance development in C. auris. These findings shed light on the multifaceted interplay between genomic diversity, SLs, and drug resistance in this emerging fungal pathogen.

Nucleic-Acid-Based Molecular Fungal Diagnostics: A Way to a Better Future.

The world has seen a tremendous increase in the number of fungal infections during the past two decades. Recently, the World Health Organisation released the pathogen priority list for fungal infections, signifying the importance of these infections in the fields of research and public health. Microbiology laboratories demand an upgrade in the diagnostic system to keep up with the increased burden of these infections. Diagnosis of fungal infections using conventional techniques has always faced limitations in terms of specificity, sensitivity, and turnaround time. Although these methods are the core pillars of the diagnosis, there is an increased need for molecular approaches. Molecular techniques have revolutionised the field of fungal diagnostics. The diverse array of molecular techniques, including techniques like Polymerase Chain Reaction (PCR), have emerged as a cornerstone in fungal diagnostics. Molecular techniques have transformed fungal diagnostics, providing powerful tools for the rapid and accurate identification of pathogens. As these technologies continue to evolve, their integration into routine clinical practice holds the promise of improving patient outcomes through timely and targeted antifungal interventions. This review will cover the molecular approaches involved in fungal diagnostics, moving from the basic techniques to the advanced-level nucleic-acid-based molecular approaches providing a high throughput and decreased turnaround time for the diagnosis of serious fungal infections.

Polymorphisms in Innate and Adaptive Immune Genes in Subjects with Allergic Bronchopulmonary Aspergillosis Complicating Asthma.

Innate and adaptive immunity play a crucial role in allergic bronchopulmonary aspergillosis (ABPA) pathogenesis. We performed next-generation sequencing using the Illumina TruSight One panel (4,811 human disease-associated genes, at least 20 × coverage) and selected 22 known immune genes (toll-like receptors (TLRs), C-type lectin, interleukin-4 receptor, and others). We included ABPA (n = 18), asthma without ABPA (n = 12), and healthy controls (n = 8). We analyzed 3011 SNPs from 22 genes and identified 145 SNPs (13 genes) that were present only in the disease groups and absent in controls. The SNP frequency overall was significantly higher in ABPA than in asthmatics (89/145 [61.4%] vs. 56/145 [38.6%], p = 0.0001). The SNP frequency in the TLR10 gene was also significantly higher in ABPA than in asthma (p = 0.017). Association analysis further revealed three genes having significant associations. Of these, NOS3 and HLA-DQB1 are associated with antimicrobial activity and adaptive immunity. More extensive studies are required to confirm our findings.

Revised ISHAM-ABPA working group clinical practice guidelines for diagnosing, classifying and treating allergic bronchopulmonary aspergillosis/mycoses.

BACKGROUND: The International Society for Human and Animal Mycology (ISHAM) working group proposed recommendations for managing allergic bronchopulmonary aspergillosis (ABPA) a decade ago. There is a need to update these recommendations due to advances in diagnostics and therapeutics. METHODS: An international expert group was convened to develop guidelines for managing ABPA (caused by Aspergillus spp.) and allergic bronchopulmonary mycosis (ABPM; caused by fungi other than Aspergillus spp.) in adults and children using a modified Delphi method (two online rounds and one in-person meeting). We defined consensus as ≥70% agreement or disagreement. The terms "recommend" and "suggest" are used when the consensus was ≥70% and <70%, respectively. RESULTS: We recommend screening for A. fumigatus sensitisation using fungus-specific IgE in all newly diagnosed asthmatic adults at tertiary care but only difficult-to-treat asthmatic children. We recommend diagnosing ABPA in those with predisposing conditions or compatible clinico-radiological presentation, with a mandatory demonstration of fungal sensitisation and serum total IgE ≥500 IU·mL-1 and two of the following: fungal-specific IgG, peripheral blood eosinophilia or suggestive imaging. ABPM is considered in those with an ABPA-like presentation but normal A. fumigatus-IgE. Additionally, diagnosing ABPM requires repeated growth of the causative fungus from sputum. We do not routinely recommend treating asymptomatic ABPA patients. We recommend oral prednisolone or itraconazole monotherapy for treating acute ABPA (newly diagnosed or exacerbation), with prednisolone and itraconazole combination only for treating recurrent ABPA exacerbations. We have devised an objective multidimensional criterion to assess treatment response. CONCLUSION: We have framed consensus guidelines for diagnosing, classifying and treating ABPA/M for patient care and research.

Sensitivity and specificity of LDBio Aspergillus ICT lateral flow assay for diagnosing allergic bronchopulmonary aspergillosis in adult asthmatics.

BACKGROUND: Aspergillus fumigatus-specific IgG estimation is crucial for diagnosing allergic bronchopulmonary aspergillosis (ABPA). A point-of-care LDBio immunochromatographic lateral flow assay (LFA) had 0%-90% sensitivity to detect IgG/IgM antibodies against A. fumigatus. OBJECTIVE: To assess the accuracy of LDBio-LFA in diagnosing ABPA, using the modified ISHAM-ABPA working group criteria as the reference standard. The secondary objective was to compare the diagnostic performance between LDBio-LFA and A. fumigatus-specific IgG (cut-offs, 27 and 40 mgA/L), using a multidisciplinary team (blinded to A. fumigatus-IgG and LDBio-LFA results) diagnosis of ABPA as the reference standard. METHODS: We prospectively enrolled adult subjects with asthma and ABPA. We performed the LDBio-LFA per the manufacturer's recommendations. We used the commercially available automated fluorescent enzyme immunoassay for measuring serum A. fumigatus-specific IgG. We used the same serum sample to perform both index tests. The tests were performed by technicians blinded to the results of other tests and clinical diagnoses. RESULTS: We included 123 asthmatic and 166 ABPA subjects, with a mean ± SD age of 37.4 ± 14.4 years. Bronchiectasis and high-attenuation mucus were seen in 93.6% (146/156) and 24.3% (38/156) of the ABPA subjects. The sensitivity and specificity of LDBio-LFA in diagnosing ABPA were 84.9% and 82.9%, respectively. The sensitivity of serum A. fumigatus-specific IgG ≥27 mgA/L was 13% better than LDBio-LFA, with no difference in specificity. There was no significant difference in sensitivity and specificity between LDBio-LFA and serum A. fumigatus-IgG ≥40 mgA/L. CONCLUSION: LDBio-LFA is a valuable test for diagnosing ABPA. However, a negative test should be confirmed using an enzyme immunoassay.

Incidence and prevalence of chronic pulmonary aspergillosis in patients with post-tuberculosis lung abnormality: Results from a community survey in North India.

BACKGROUND: Post-tuberculosis lung abnormality (PTLA) is the most common risk factor for developing chronic pulmonary aspergillosis (CPA). However, the prevalence and incidence of CPA in PTLA patients in India remain unknown. OBJECTIVES: We aimed to ascertain the incidence and prevalence of CPA in subjects with PTLA. METHODS: We identified a cohort of pulmonary tuberculosis who completed anti-tuberculosis therapy (ATT) before November 2019 from the records of the 12 tuberculosis treatment centers attached to the national program. We recorded the clinical and demographic details. We performed computed tomography (CT) of the chest and estimated serum A. fumigatus-specific IgG. We categorised subjects as PTLA with or without CPA using a composite of clinical, radiological, and microbiological features. We resurveyed the subjects at 6 months (or earlier) for the presence of new symptoms. We calculated the prevalence and the incidence rate (per 100-person years) of CPA. RESULTS: We included 117 subjects with PTLA, with a median of 3 years after ATT completion. Eleven subjects had CPA in the initial survey, and one additional case developed CPA during the second survey. The prevalence of CPA in PTLA subjects was 10.3% (12/117). The total observation period was 286.7 person-years. The median (interquartile range) time to develop CPA after ATT completion was 12.5 (5-36.7) months. We found the CPA incidence rate (95% confidence interval) of 4.2 (1.8-6.5) per 100-person years. CONCLUSION: Chronic pulmonary aspergillosis complicates 10% of PTLA subjects after successful outcomes with ATT. Four new CPA cases may develop per 100-persons years of observation after ATT completion. We suggest screening patients with PTLA who develop new symptoms for CPA.

Mucormycosis: Cytomorphological Spectrum in Fine-Needle Aspiration Cytology.

Background: Mucormycosis is a fungal infection that can affect multiple organs. The role of fine-needle aspiration cytology (FNAC) in its diagnosis is not well documented. Aim: The objective of this study was to describe the detailed cytomorphologic features of mucormycosis on FNAC samples. Materials and Methods: A retrospective analysis of all cases diagnosed as mucormycosis on FNAC between January 2014 and July 2021 was performed for detailed cytomorphological evaluation and correlation to clinical data and microbiological studies wherever available. FNA was computed tomography-guided (n = 38), ultrasonography-guided (n = 31) or palpation-guided (n = 12), and slides were reviewed in two cases. Results: A total of 83 cases of mucormycosis were evaluated. An immunocompromised setting was observed in 48 cases. The most common site of FNA was the lung (n = 57) followed by liver, soft tissue, palate, mediastinum, orbital/ocular region, and lymph node. Isolated renal involvement, a unique feature, was seen in seven cases. The aspirates were necrotic to pus-like or blood-mixed particulate. Broad, nonseptate, foldable, ribbon-like fungal hyphae showing right-angled branching were seen. The tissue reaction was predominantly necro-inflammatory (n = 36), bland necrotic (n = 22), mixed inflammatory (n = 16), suppurative (n = 5), necrotizing granulomatous (n = 3), and granulomatous (n = 1). Immunocompromised patients showed mixed inflammatory responses more frequently. Fungal culture was positive for Rhizopus species in 2/13 cases and molecular testing in two additional cases corresponding to Rhizopus and Syncephalastrum spp. Conclusion: FNA provides quick and conclusive diagnosis of mucormycosis from varied anatomic sites enabling prompt institution of therapy. The tissue response is variable and to some extent dependent on the immune status of the patient.

A promising antifungal lipopeptide from Bacillus subtilis: its characterization and insight into the mode of action.

Emerging resistance of fungal pathogens and challenges faced in drug development have prompted renewed investigations into novel antifungal lipopeptides. The antifungal lipopeptide AF3 reported here is a natural lipopeptide isolated and purified from Bacillus subtilis. The AF3 lipopeptide's secondary structure, functional groups, and the presence of amino acid residues typical of lipopeptides were determined by circular dichroism, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The lipopeptide's low minimum inhibitory concentrations (MICs) of 4-8 mg/L against several fungal strains demonstrate its strong antifungal activity. Biocompatibility assays showed that ~ 80% of mammalian cells remained viable at a 2 × MIC concentration of AF3. The treated Candida albicans cells examined by scanning electron microscopy, transmission electron microscopy, and atomic force microscopy clearly showed ultrastructural alterations such as the loss of the cell shape and cell membrane integrity. The antifungal effect of AF3 resulted in membrane permeabilization facilitating the uptake of the fluorescent dyes-acridine orange (AO)/propidium iodide (PI) and FUN-1. Using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 4-(2-[6-(dioctylamino)-2-naphthalenyl] ethenyl)-1-(3-sulfopropyl) pyridinium inner salt (di-8-ANEPPS), we observed that the binding of AF3 to the membrane bilayer results in membrane disruption and depolarization. Flow cytometry analyses revealed a direct correlation between lipopeptide activity, membrane permeabilization (~ 75% PI uptake), and reduced cell viability. An increase in 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence demonstrates endogenous reactive oxygen species production. Lipopeptide treatment appears to induce late-stage apoptosis and alterations to nuclear morphology, suggesting that AF3-induced membrane damage may lead to a cellular stress response. Taken together, this study illustrates antifungal lipopeptide's potential as an antifungal drug candidate. KEY POINTS: • The studied lipopeptide variant AF3 displayed potent antifungal activity against C. albicans • Its biological activity was stable to proteolysis • Analytical studies demonstrated that the lipopeptide is essentially membranotropic and able to cause membrane dysfunction, elevated ROS levels, apoptosis, and DNA damage.

Immune and metabolic perturbations in COVID-19-associated pulmonary mucormycosis: A transcriptome analysis of innate immune cells.

BACKGROUND AND OBJECTIVES: The mechanisms underlying COVID-19-associated pulmonary mucormycosis (CAPM) remain unclear. We use a transcriptomic analysis of the innate immune cells to investigate the host immune and metabolic response pathways in patients with CAPM. PATIENTS AND METHODS: We enrolled subjects with CAPM (n = 5), pulmonary mucormycosis (PM) without COVID-19 (n = 5), COVID-19 (without mucormycosis, n = 5), healthy controls (n = 5) without comorbid illness and negative for SARS-CoV-2. Peripheral blood samples from cases were collected before initiating antifungal therapy, and neutrophils and monocytes were isolated. RNA sequencing was performed using Illumina HiSeqX from monocytes and neutrophils. Raw reads were aligned with HISAT-2 pipeline and DESeq2 was used for differential gene expression. Gene ontology (GO) and metabolic pathway analysis were performed using Shiny GO application and R packages (ggplot2, Pathview). RESULTS: The derangement of core immune and metabolic responses in CAPM patients was noted. Pattern recognition receptors, dectin-2, MCL, FcRγ receptors and CLEC-2, were upregulated, but signalling pathways such as JAK-STAT, IL-17 and CARD-9 were downregulated; mTOR and MAP-kinase signalling were elevated in monocytes from CAPM patients. The complement receptors, NETosis, and pro-inflammatory responses, such as S100A8/A9, lipocalin and MMP9, were elevated. The major metabolic pathways of glucose metabolism-glycolysis/gluconeogenesis, pentose phosphate pathway, HIF signalling and iron metabolism-ferroptosis were also upregulated in CAPM. CONCLUSIONS: We identified significant alterations in the metabolic pathways possibly leading to cellular iron overload and a hyperglycaemic state. Immune responses revealed altered recognition, signalling, effector functions and a pro-inflammatory state in monocytes and neutrophils from CAPM patients.

Opportunistic microsporidiosis unveiled by fine-needle aspiration cytology of cervical lymph node with literature review.

Microsporidia are highly specialized obligate intracellular organisms closely related to fungi, traditionally linked to diarrheal diseases in acquired immunodeficiency syndrome patients. Over the past two decades, an increasing incidence of extraintestinal infections affecting various organ systems, especially in immunocompromised individuals, has been observed. The report presents a unique case of lymph node microsporidiosis in a 38-year-old male, positive for human immunodeficiency virus, with coinfections of hepatitis B and C. Fine-needle aspiration cytology (FNAC) from cervical lymph node yielded pus-like, necrotic material with periodic acid-Schiff stained smear uncovering small round to oval spores on microscopy suspicious for microsporidia. Based on polymerase chain reaction and sequencing done with aspiration material, the causative agent was identified as Vittaforma corneae. This rare encounter highlights the significance of recognizing unique morphological characteristics of infectious organisms and employing appropriate ancillary techniques for precise identification. The case underscores the crucial role of FNAC in diagnosing opportunistic infections involving the lymph nodes and the growing significance of molecular tests for specific pathogen confirmation.

Prevalence of Aspergillus sensitization and Allergic Bronchopulmonary Aspergillosis in bronchial asthma: A systematic review of Indian studies.

Background: The prevalence of allergic bronchopulmonary aspergillosis (ABPA) in Indian asthmatic patients remains unknown. We systematically reviewed the literature for estimating the prevalence of Aspergillus sensitization (AS) and ABPA in Indian subjects with bronchial asthma. Methods: We searched the PubMed and Embase databases for studies from India reporting the prevalence of AS or ABPA in at least 50 asthmatics. The primary outcome of our study was to assess the prevalence of ABPA. The secondary outcomes were to evaluate the prevalence of AS in asthma and ABPA in Aspergillus-sensitized asthma. We pooled the prevalence estimates using a random effects model and examined the factors influencing the prevalence using multivariate meta-regression. Results: Of the 8,383 records retrieved, 34 studies with 14,580 asthmatics met the inclusion criteria. All the studies were from tertiary centers. The pooled prevalence of ABPA in asthmatics (26 studies; 5,554 asthmatics) was 16.2% [95% confidence interval (CI), 12.5-20.4]. The pooled prevalence of AS in asthma (29 studies; 13,405 asthmatics) was 30.9% (95% CI, 25.3-36.6), while the prevalence of ABPA in AS (20 studies; 1,493 asthmatics) was 48.2% (95% CI, 39.6-56.8). Meta-regression identified studies published after 2009 (OR 1.14; 95% CI, 1.02-1.28) and studies with severe asthmatics (OR 1.12; 95% CI, 1.00-1.26) as the only factors associated with higher ABPA prevalence. Conclusions: There is a high prevalence of ABPA in Indian asthmatic subjects at tertiary centers, underscoring the need for screening all asthmatic subjects in special asthma and chest clinics for ABPA.

Cladosporium halotolerans: Exploring an Unheeded Human Pathogen.

BACKGROUND: Cladosporium halotolerans is a saprobic fungus, rarely implicated in human infections. The identification is challenging due to non-specific phenotypic features. OBJECTIVE: To decipher clinical spectrum, microbiological and susceptibility profile of clinical and environmental isolates of Cladosporium halotolerans. METHOD: All the isolates identified as Cladosporium halotolerans deposited in National Culture Collection for Pathogenic Fungi (NCCPF), Postgraduate Institute of Medical Education and Research, Chandigarh, India were revived. Phenotypic and molecular characterization targeting internal transcribed spacer (ITS) region of ribosomal DNA, large subunit of ribosomal DNA (LSU; NL1 and NL4), actin (ACT) and beta-tubulin (TUB) was done. Scanning electron microscopy (SEM) was performed to determine any phenotypic variations. Antifungal susceptibility testing (AFST) was carried out for eight antifungal agents as per CLSI M38 Ed3 guidelines. We also performed systematic literature review of all the cases of Cladosporium halotolerans reported till date. RESULTS: A total of four isolates (clinical, n = 3; soil, n = 1) identified as Cladosporium halotolerans were included in the study. The clinical sites were skin, maxillary tissue and nail. All patients were apparently immunocompetent, and history of trauma was recorded in one patient. All patients improved on antifungal therapy. The cultures revealed growth of black mycelial fungus and microscopic examination demonstrated dematiaceous septate hyphae with erect conidiophores and conidia in branched acropetal chains. Based on molecular methods, all the four isolates were identified as C. halotolerans. SEM revealed no variation in length and width of the conidia, conidiophores, ramoconidium and hyphae among the isolates. All molecular targets, such as ITS region, LSU (partially sequenced), ACT and TUB were able to differentiate the isolates. Minimum inhibitory concentrations for antifungals were: triazoles (0.12-2 µg/ml), amphotericin B (4 µg/ml) and echinocandins (2-8 µg/ml). CONCLUSION: We report role of the rarely isolated dematiaceous fungus, C. halotolerans, in causing human infections. The study emphasizes the role of molecular methods in precisely identifying these species. Triazoles are more active against these black fungi compared to polyenes or echinocandins.