Insights into Changing Dermatophyte Spectrum in India Through Analysis of Cumulative 161,245 Cases Between 1939 and 2021.

Dermatophytosis is one of the most common superficial infections of the skin affecting nearly one-fifth of the world population at any given time. With nearly 30% of worldwide terbinafine-resistance cases in Trichophyton mentagrophytes/Trichophyton interdigitale and Trichophyton rubrum reported from India in recent years, there is a significant burden of the emerging drug resistance epidemic on India. Here, we carry out a comprehensive retrospective analysis of dermatophytosis in India using 1038 research articles pertaining to 161,245 cases reported from 1939 to 2021. We find that dermatophytosis is prevalent in all parts of the country despite variable climatic conditions in different regions. Our results show T. rubrum as the most prevalent until 2015, with a sudden change in dermatophyte spectrum towards T. mentagrophytes/T. interdigitale complex since then. We also carried out an 18S rRNA-based phylogenetics and an average nucleotide identity-and single nucleotide polymorphism-based analysis of available whole genomes and find very high relatedness among the prevalent dermatophytes, suggesting geographic specificity. The comprehensive epidemiological and phylogenomics analysis of dermatophytosis in India over the last 80 years, presented here, would help in region-specific prevention, control and treatment of dermatophyte infections, especially considering the large number of emerging resistance cases.

Burden of Chronic Dermatophytosis in a Tertiary Care Hospital: Interaction of Fungal Virulence and Host Immunity.

Dermatophytosis is caused by keratinophilic dermatophytes and affects the superficial skin and its appendages. The nature of infection and response to treatment is influenced by host-pathogen factors like duration and severity of disease, prior drug history and type of causative organism. In our study, the burden of dermatophytosis affecting glabrous skin saw a rise in recalcitrant and reinfection cases with only 1.6% achieving complete cure. Chronicity of dermatophytic infection was reflected in the high serum IgE levels and immediate hypersensitivity reactions. Hence, it becomes pertinent for clinicians to identify the non-responders and modify therapy to achieve clinical cure with fungal clearance confirmed by mycological tools.

Possible Role of Trichophytin Antigen in Inducing Impaired Immunological Clearance of Fungus in Onychomycosis.

The immunology of onychomycosis is poorly understood. Th1 and Th17 are the principal effector cells responsible for protective immunity against fungi, while it is assumed that Th2 responses are associated with deleterious effects. The study was conducted to appraise the role of interleukin-6 (IL-6), transforming growth factor ß (TGF-ß) and immunoglobulin E (IgE) in onychomycosis patients and to study skin reactivity to trichophytin antigen in them. Serum samples of 60 cases of chronic onychomycosis and 30 healthy controls were assayed for serum IgE, IL-6 and TGF-ß levels using specific immunoassay kits; 0.01 ml of trichophytin antigen, Candida antigen and phosphate-buffered saline using separate syringes were injected intradermal at three independent sites of the forearm in cases and controls. Serum IL-6 levels were significantly lower in cases as compared to controls, while serum TGF-ß levels in both cases and controls were comparable. Serum IgE levels in cases were significantly higher when compared with controls. Thirty-eight patients showed immediate hypersensitivity response to trichophytin antigen, while none showed delayed hypersensitivity reaction to trichophytin antigen. Constant fungal antigenic stimuli induce a state of anergy as indicated by low serum IL-6 levels and the absence of delayed hypersensitivity reaction to trichophytin antigen in cases, leading to chronicity of infection. High total IgE may indicate a high probability of prior fungal sensitization.

Genotyping and In Vitro Antifungal Susceptibility Testing of Fusarium Isolates from Onychomycosis in India.

Onychomycosis refers to fungal infection of the nail and is commonly caused by dermatophytes, while yeasts and non-dermatophytic molds (NDM) are increasingly recognized as pathogens in nail infections. The present study was done to delineate molecular epidemiology of Fusarium onychomycosis in India. Five hundred nail samples of Indian patients clinically suspected of onychomycosis were subjected to direct microscopy and fungal culture. Representative Fusarium isolates were further identified to species level by multi-locus sequencing for internal transcribed spacer, translation elongation factor 1 alpha (tef1-α) and RNA polymerase II subunit (rpb2) regions (primer pairs: ITS1/ITS4, EF1/EF2, 5f2/7cr, respectively). These representative strains were also tested for in vitro antifungal susceptibility by the broth microdilution method. Members of the genus Fusarium proved to be the most common NDM responsible for onychomycosis. The Fusarium spp. responsible for onychomycosis belonged to the Fusarium solani species complex (F. keratoplasticum and F. falciforme) and Fusarium fujikuroi species complex (F. proliferatum, F. acutatum and F. sacchari). Antifungal susceptibility results indicated that amphotericin B was the most effective antifungal across all isolates (MIC ranging 0.5-2 mg/L), followed by voriconazole (MIC ranging 1-8 µg/ml). However, a large variation was shown in susceptibility to posaconazole (MIC ranging 0.5 to >16 µg/ml). To conclude, we identified different Fusarium spp. responsible for onychomycosis in India with variation within species in susceptibility to antifungal agents, showing that fusariosis requires correct and prompt diagnosis as well as antifungal susceptibility testing.

Association of variants in BAT1-LTA-TNF-BTNL2 genes within 6p21.3 region show graded risk to leprosy in unrelated cohorts of Indian population.

Host immune response against Mycobacterium leprae plays an important role in providing resistance to infection and disease progression. Genome-wide linkage and association studies suggest the possibility of multiple risk loci within HLA (6p21.3) region. Any systematic study of relevance within the histocompatibility complex of importance in host immune response would be pertinent because of non-replication of the known loci and unavailable information on some of the unexplored genes and regions. A systematic scan was performed of the selected region involving LTA-TNF-LTB genes within 6p21.3 with a resolution of 1SNP/127 bp; and the SNPs in flanking BAT1, NFKBIL and BTNL2-DRA genes on the basis of their tag status or their presence in promoter/exonic regions with MAF of >5%. Nine SNPs located in BAT1, LTA, TNF genes and BTNL2-DRA interval showed strong association with leprosy susceptibility in two independent sets of North Indian population which was replicated in a geographically distinct East Indian population. Conditional logistic regression showed at least one functional SNP remaining significant in each gene, suggesting an independent role of each of the disease associated SNPs. In vitro reporter assay revealed that two SNPs located at BAT1 promoter and 13 kb upstream to LTA gene affected the transcription factor binding site, hence the gene expression. We unravel the role of unexplored immunologically important genes, BAT1 and BTNL2, in addition to known LTA and TNF genes, and the haplotypes of the significantly associated SNPs therein, to understand susceptibility to the disease, leprosy and its differential severity.

Genetic variations and interactions in anti-inflammatory cytokine pathway genes in the outcome of leprosy: a study conducted on a MassARRAY platform.

BACKGROUND: Mycobacterium leprae is the etiologic pathogen that causes leprosy. The outcome of disease is dependent on the host genetic background. METHODS: We investigated the association of 51 single-nucelotide polymorphisms (SNPs) in anti-inflammatory cytokines (IL-10, TGFB1, IL-6, IL-4, and IL-13) and receptors (IL-10RA, IL-10RB, TGFBR1, TGFBR2, IL-6R, IL-4R, IL-5RA, IL-5RB, and IL-13RA1) with susceptibility to leprosy in a case-control study from New Delhi in northern India. This was followed by replication testing of associated SNPs in a geographically distinct and unrelated population from Orissa in eastern India. The functional potential of SNPs was established with in vitro reporter assays. RESULTS: Significant associations (P < .05) were observed for 8 polymorphisms (rs1800871, rs1800872, and rs1554286 of IL-10; rs3171425 and rs7281762 of IL-10RB; rs2228048 and rs744751 of TGFBR2; and rs1800797 of IL-6) with leprosy. This association was replicated for 4 SNPs (rs1554286 of IL-10, rs7281762 of IL-10RB, rs2228048 of TGFBR2, and rs1800797 of IL-6). The interaction study revealed a significantly greater association with leprosy risk than was obtained for any SNP individually. CONCLUSIONS: This study provides an interesting insight on the cumulative polygenic host component that regulates leprosy pathogenesis.

Juvenile, insulin-dependent diabetes mellitus, type 1-related dermatoses.

Juvenile insulin-dependent diabetes mellitus type 1 (IDDM) is a well-recognized worldwide entity, the significance of which has increased because of its recent upsurging trends, warranting attention on variety of its clinical expressions, in particular, pertaining to skin, an aspect seldom taken cognizance of. Hence an endeavour to recap the related dermatoses, such as limited joint mobility syndrome including sclerodermoid (scleroderma-like) changes, xerosis, necrobiosis lipoidica diabeticorum, granuloma annulare, diabetic foot syndrome, has been made. Complexities relating to the recently explored issues of atopic dermatitis and drug hypersensitivity syndrome have also been covered adequately. In addition, the current concepts of the physiopathology of type 1 diabetes-related dermatoses are briefly recapitulated for clarity.

Gene expression profiling of protease and non-protease genes in Trichophyton mentagrophytes isolates from dermatophytosis patients by qRT-PCR analysis.

Trichophyton mentagrophytes secretes Metallocarboxypeptidase A and B of the M14 family as endoproteases and exoprotease. T. mentagrophytes produce Metalloprotease 3 and 4 which degrades the protein into the short peptides and amino acids. To understand the host fungal relationship and identification of such genes expressed during infection is utmost important. T. mentagrophytes encodes some proteins which are associated with the glyoxylate cycle. The glyoxylate cycle enzymes have been involving in virulence of dermatophytes and their up-regulation during dermatophytes growth on keratin. On comparing the expression level of virulence protease and non-protease genes, we observed, among exoprotease protease genes, Metallocarboxypeptidase B was strongly up regulated (134.6 fold high) followed by Metallocarboxypeptidase A (115.6 fold high) and Di-peptidyl-peptidases V (10.1 fold high), in dermatophytic patients as compared to ATCC strain. Furthermore, among endoprotease, Metalloprotease 4 was strongly up regulated (131.6 fold high) followed by Metalloprotease 3 (16.7 fold high), in clinical strains as compared to T. mentagrophytes ATCC strain. While among non-protease genes, Citrate Synthase was highly expressed (118 fold high), followed by Isocitrate Lyase (101.6 fold high) and Malate Synthase (52.9 fold high). All the studied virulence genes were considered the best suitable ones by geNorm, Best keeper, Norm Finder and Ref finder.

Whole genome sequences of two Trichophyton indotineae clinical isolates from India emerging as threats during therapeutic treatment of dermatophytosis.

In the current study, we report the genome sequence of two different clinical isolates from India, Trichophyton indotineae UCMS-IGIB-CI12 and Trichophyton indotineae UCMS-IGIB-CI14. The resulting genome assembly achieved a 143-fold coverage in 824 contigs for T. indotineae UCMS-IGIB-CI12 and a 136-fold coverage in 904 contigs for T. indotineae UCMS-IGIB-CI14. Both the clinical isolates contain a c.1342G>A mutation corresponding to Ala448Thr amino acid substitution in erg1 and exhibit an intermittent drug response to terbinafine. Comparative genomics analysis with available genomes of Trichophyton interdigitale/Trichophyton mentagrophytes species complex revealed a similar genome architecture and identified large number of genes associated with virulence and pathogenicity, namely, lipases, proteases, LysM domain-containing factors, carbon metabolism enzymes and cytochrome P450 enzymes, in all the genomes. An analysis of single amino acid polymorphisms (SAPs) in the protein sequences of subtilisin and lipase enzyme families identified a higher frequency of SAPs in functionally important proteins, Sub3 and Sub6 and their possible use in multilocus phylogenetic analysis of T. interdigitale/T. mentagrophytes species complex. The whole genome sequences of T. indotineae clinical isolates provided in this report will, hence, serve as a key reference point for investigation of clinical strains and emerging drug resistance among dermatophytes originating from different parts of the world.

Correction to: Whole genome sequences of two Trichophyton indotineae clinical isolates from India emerging as threats during therapeutic treatment of dermatophytosis.

[This corrects the article DOI: 10.1007/s13205-021-02950-1.].

Terbinafine resistance in dermatophytes: Time to revisit alternate antifungal therapy.

Resistant superficial dermatophytic infections of the skin and its appendages have emerged as a major health problem in India. Mutations in Squalene epoxidase gene have led to increasing incidence of resistance to terbinafine in dermatophytic isolates. We examined six patients with recalcitrant dermatophytosis attending Dermatology OPD at a tertiary care hospital and demonstrated terbinafine resistance by molecular method. Immediate hyperitivity (IH) reaction to Trichophytin antigen was highlighted in these patients. The patients were treated with alternate antifungals after demonstration of resistance to terbinafine based on the antifungal susceptibility testing (AFST). On follow up the patients responded well to the substitute but the duration of therapy had to be prolonged beyond six weeks.

T helper 1 to T helper 2 shift in cytokine expression: an autoregulatory process in superantigen-associated psoriasis progression?

Psoriasis is an inflammatory skin disorder characterized by increased activation of CD4(+) T lymphocytes, and systemic and local overexpression of pro-inflammatory cytokines such as interleukin 2 (IL-2), gamma interferon (IFN-gamma), IL-6 and tumour necrosis factor alpha, indicating that immunopathogenesis of the disease is T helper 1 (Th1) mediated. Several studies suggest a pivotal role of bacterial superantigens in the initiation and/or exacerbation of this illness. This study was conducted to assess the systemic Th1/Th2 imbalance in Indian psoriasis patients presenting with variable duration of disease by studying systemic superantigen-stimulated peripheral blood mononuclear cell (PBMC) cytokine expression. PBMCs were isolated and stimulated in vitro with superantigens (streptococcal pyrogenic exotoxin A and staphylococcal enterotoxin B), and the cytokines released (IFN-gamma for a Th1 response, and IL-4 and IL-10 for a Th2 response) were assayed. In contrast to controls, psoriasis patients in the early course of disease were characterized by significantly increased expression of the pro-inflammatory cytokine IFN-gamma, whilst a shift towards IL-10 secretion (Th2 response) was observed in those presenting with increased duration of disease. These observations suggest a possible shift from a Th1 to a Th2 cytokine response with superantigen-associated progression for the duration of psoriasis, perhaps as an adaptive process by the immune system in an attempt to downregulate abnormal inflammatory Th1 immune responses.

Laboratory-based epidemiological study of superficial fungal infections.

Dermatophytosis continues to be a worldwide problem, constituting a large bulk of cases attending the dermatology outpatient's department in tropical countries. Variable climatic conditions and multiple etiological agents, whose individual prevalence varies with time, prompted an attempt to define the current pattern and etiologic prevalence in our locality, and compare them with earlier studies from different centers. Of consenting patients clinically diagnosed to have superficial fungal infections, 1975 were investigated in the laboratory. All the specimens collected from patient skin, hair or nails were subjected to direct microscopy examination in 10% potassium hydroxide (KOH) and fungal culture. Confirmed diagnosis was made only if specimen was KOH and/or culture positive. Male : female ratio was 1.65 : 1. Tinea corporis (21.4%), onychomycoses (14.7%) and tinea capitis (6.2%) were the most common laboratory-confirmed infections. Only 909 patients (47%) out of 1035 clinically suspected patients had evidence of fungal infection by either microscopy and/or culture. Of these 909 patients, 787 (86.5%) were both KOH and culture positive, 25 (2.7%) were KOH negative and culture positive, while 97 (10.6%) were KOH positive but culture negative. In 1051 patients (53%), no evidence of fungal infection was seen. Trichophyton rubrum was the most commonly isolated dermatophyte (55.2%) followed by Trichophyton mentagrophytes (19.6%) and Trichophyton tonsurans (2.9%). Candida sp. accounted for 16% of all isolates. Non-dermatophyte moulds (NDM) were isolated only in patients with onychomycoses. Our study revealed a male preponderance, tinea corporis as the commonest clinical type, and dermatophytes as the commonest mycological isolates, which is in agreement with earlier studies. Relying on clinical diagnosis alone without laboratory confirmation may result in overestimation of the problem as evidenced in other studies as compared to our study. Rarely reported NDM appear to be important etiological agents of onychomycoses.

Computer assisted Objective structured clinical examination versus Objective structured clinical examination in assessment of Dermatology undergraduate students.

BACKGROUND: The assessment of dermatology undergraduates is being done through computer assisted objective structured clinical examination at our institution for the last 4 years. We attempted to compare objective structured clinical examination (OSCE) and computer assisted objective structured clinical examination (CA-OSCE) as assessment tools. AIM: To assess the relative effectiveness of CA-OSCE and OSCE as assessment tools for undergraduate dermatology trainees. METHODS: Students underwent CA-OSCE as well as OSCE-based evaluation of equal weightage as an end of posting assessment. The attendance as well as the marks in both the examination formats were meticulously recorded and statistically analyzed using SPSS version 20.0. Intercooled Stata V9.0 was used to assess the reliability and internal consistency of the examinations conducted. Feedback from both students and examiners was also recorded. RESULTS: The mean attendance for the study group was 77% ± 12.0%. The average score on CA- OSCE and OSCE was 47.4% ± 19.8% and 53.5% ± 18%, respectively. These scores showed a mutually positive correlation, with Spearman's coefficient being 0.593. Spearman's rank correlation coefficient between attendance scores and assessment score was 0.485 for OSCE and 0.451 for CA-OSCE. The Cronbach's alpha coefficient for all the tests ranged from 0.76 to 0.87 indicating high reliability. LIMITATIONS: The comparison was based on a single batch of 139 students. Such an evaluation on more students in larger number of batches over successive years could help throw more light on the subject. CONCLUSIONS: Computer assisted objective structured clinical examination was found to be a valid, reliable and effective format for dermatology assessment, being rated as the preferred format by examiners.

Malassezia Yeast and Cytokine Gene Polymorphism in Atopic Dermatitis.

INTRODUCTION: Atopic Dermatitis (AD) is a recurrent chronic condition associated with microorganism and their interaction with the susceptible host. Malassezia yeast is a known commensal which is thought to provoke the recurrent episodes of symptoms in atopic dermatitis patients. Malassezia immunomodulatory properties along with defective skin barrier in such host, results in disease manifestation. Here, we studied Single Nucleotide Polymorphism (SNP) in IL10 and IFN γ genes of the host and its relation with susceptibility to Malassezia infection. AIM: To isolate Malassezia yeast from AD patients and compare the genetic susceptibility of the host by correlating the cytokine gene polymorphism with the control subjects. MATERIALS AND METHODS: Study was conducted from January 2012 to January 2013. It was a prospective observational study done in Department of Microbiology and Department of Dermatology and Venereology in University College of Medical Sciences and GTB Hospital, Delhi. Sample size comprised of 38 cases each of AD. Skin scrapings were used for fungal culture on Sabouraud Dextrose Agar (SDA) and Modified Dixon Agar (MDA) and isolated were identified as per conventional phenotypic methods. Genomic DNA was extracted from blood samples collected from all study subjects. Cytokine genotyping was carried out by Amplification Refractory Mutations System- Polymerase Chain Reaction (ARMS-PCR) with sequence specific primers. Three SNPs (IL10-1082A/G; IL10-819/592C/T; IFN-γ+874A/T) in two cytokine genes were assessed in all the patients and healthy controls. STATISTICAL ANALYSIS: Chi-Square Test or Fisher's-Exact Test and Bonferroni's correction. RESULTS: In AD group, Malassezia yeasts were cultured in 24 out of 38 samples and thus the identification rate was 63.1 percent as compared to healthy group, 52.6 percent (20/38). Significant difference in allele, or genotype distribution were observed in IL10-819/592C/T and IFN-γ+874A/T gene polymorphism in AD group. CONCLUSION: Higher isolation rate in cases as compared to control group highlights the implication of Malassezia in AD. Association between specific cytokine gene polymorphism and clinical outcome was found to be significant in study group. The result of cytokine gene polymorphism in the present study demonstrated susceptibility of host to Malassezia infection.

Fine-needle aspiration cytology of lepromatous leprosy.

A prospective study correlating cytopathology with clinical morphology and histopathology in 22 patients with lepromatous leprosy was performed. Aspirates were taken from skin lesions in all patients. Lymph node aspirates were also performed in four patients with lymphadenopathy. Fine-needle aspirates yielded sufficient cellular material with excellent preservation of morphological detail. Diagnosis and correlation with bacillary index, clinical and histopathological findings was possible in all patients. In addition, the two patterns, partial and diffuse, of lymph node involvement could be recognized. Fine-needle aspiration cytology is a simple method for the laboratory assessment of leprosy.

Soluble interleukin-2 receptors: levels in leprosy, and during and after type 1 (lepra) and type 2 (ENL) reactions.

Twenty-five patients with Type 1 (lepra) and Type 2 (ENL) reactions, were assayed for SIL-2R in serum--before and after treatment for their acute condition--and the results were compared with 10 normal healthy adults and 20 patients of leprosy per se. Classification of each subject into different leprosy groups, and into various types and subtypes of reactions, was done according to standard criteria, prior to inclusion into the study. Detailed statistical evaluation of the data revealed significantly higher levels of SIL-2R in all leprosy patients, as compared to normal controls, with higher levels in the multibacillary groups as compared to the paucibacillary group. SIL-2Rs appeared higher in Type 1 upgrading reaction than in other forms of reaction, though this was not statistically significant. There was no significant change in levels following treatment and clinical remission.