Sambit Nath Bhattacharya

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 0.5–2xa0mg/L), followed by voriconazole (MIC ranging 1–8xa0µg/ml). However, a large variation was shown in susceptibility to posaconazole (MIC ranging 0.5 to >16xa0µg/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.

Alterations in T-lymphocyte sub-set profiles and cytokine secretion by PBMC of systemic lupus erythematosus patients upon in vitro exposure to organochlorine pesticides.

Chronic exposure to organochlorine pesticides (OCP) has been suspected of causing immunoregulatory abnormalities that eventually lead to development and progression of Systemic Lupus Erythematosus (SLE), but the role of these non-genetic stimuli has remained poorly understood. The objectives of the study were to quantify the levels of different OCP residues in the blood of SLE patients and to study the effects of in vitro treatment of peripheral blood mononuclear cells (PBMC) from these patients and healthy controls with OCP. Levels of different OCP residues in the blood were measured by gas-liquid chromatography. Isolated PBMC were treated in vitro with hexachlorocyclohexane (HCH), o,p’-dichlorodiphenyltrichloroethane (DDT), or phytohemagglutinin-M (PHA-M) for 72u2009h, then stained with different dye-labeled monoclonal antibodies to analyze alterations in T-lymphocytes using flow cytometry. Levels of different TH1 and TH2 cytokines were also estimated by ELISA. Significantly higher levels of p,p’-DDE and β-HCH were detected in the blood of SLE patients than in healthy controls. HCH exposure markedly increased the percentages of CD3+CD4+ T-lymphocytes and expression of CD45RO+ on CD4+ and CD8+ T-lymphocytes, but decreased CD4+CD25+ T-lymphocytes in SLE patients. DDT exposure increased the percentages of CD3+CD4+ T-lymphocytes and decreased those of CD4+CD25+ T-lymphocytes in SLE patients as compared to healthy controls. No significant responsiveness of patient PBMC to PHA-M stimulation was observed indicating suppression of T-lymphocytes by these OCP. Further, both HCH and DDT decreased the levels of IL-2 and IFNγ but had no effect on IL-4 levels in SLE patients. DDT also increased significantly the levels of IL-10 in patients. It is likely that higher levels and prolonged durations of exposure to HCH and DDT may significantly influence T-lymphocyte sub-sets and cytokine expression in vivo that could lead to the development or exacerbation of SLE.

Clinicopathologic spectrum of cutaneous tuberculosis: a retrospective analysis of 165 Indians.

Background:Although cutaneous tuberculosis (CTB) is well described, it is wise to periodically revisit the prevailing clinical and epidemiological features, of this somewhat uncommon entity in developed nations that is also usually neglected in national tuberculosis control programs in countries where tuberculosis is a major health problem, to maintain heightened awareness as the HIV pandemic runs its course. Methods:Medical records of 165 cases of CTB, diagnosed from 2007 to 2010, were studied. The diagnosis in each was made on the basis of well-conceived and described criteria. All patients had earlier been immunized with Bacillus Calmette–Guérin soon after birth. Patients were evaluated by routine hematology, Mantoux test, screening for HIV status in addition to fine-needle aspiration cytology, and/or histopathology of the lesion. Results:Of the 165 cases (94 males and 71 females, male:female ratio of 1.32:1; age range, 1–64), 85 were lupus vulgaris, 11 tuberculosis verrucosa cutis (paucibacillary), and 39 scrofuloderma (multibacillary). The remaining 17 were tuberculids comprising 24 lichen scrofulosorum, 3 papulonecrotic tuberculid, 2 erythema induratum, and 1 erythema nodosum, afflicting children and young adults (age range, 0–10; 21–30). Fine-needle aspiration cytology was diagnostic in 39 cases, histopathology in 117, and their combination in 9. Clinicopathologic correlation and response to antituberculosis treatment were additional diagnostic adjuncts. Conclusions:CTB has a varied clinical and morphological spectrum. Cytology and histopathology play a key role in its diagnosis. An additional adjunct is the clinical response to antituberculosis treatment.

Possible role of superantigens in inducing autoimmunity in pemphigus patients

The diagnostic and pathological relevance of anti‐desmoglein autoantibodies in common forms of pemphigus has been well established, and T cells have been shown to play a role in the onset and progression of these diseases. The role of superantigens in provoking polyclonal activation of T cells with many different fine specificities, possibly including autoreactive T cells and T‐cell mediated autoantibody response, is unknown. Further, abnormal T‐cell function may lead to opportunistic infections particularly with Candida. The response of T cells of pemphigus patients to recall antigens of these opportunists is not clear. The aim of this study was to investigate the in vitro response of T lymphocytes from pemphigus patients to common bacterial superantigens such as streptococcal pyrogenic exotoxin A and staphylococcal enterotoxin B, and recall antigens such as Candida antigen. Changes in CD3+CD4+ and CD3+CD8+ T‐cell sub‐populations and expression of naive/memory markers (CD45RA+/RO+) on different T cells were analyzed by flow cytometry. Significant elevation in CD3+CD4+ and expression of the memory (CD45RO+) markers on these cells was observed both in pemphigus vulgaris and pemphigus foliaceus patients, as compared to healthy controls, upon stimulation with streptococcal pyrogenic exotoxin A and staphylococcal enterotoxin B. However, only memory T cells (CD45RO+) were significantly increased upon Candida antigen stimulation. Our study suggests that CD4+ memory T lymphocytes may modulate the pathogenic autoantibody response in pemphigus patients, and also emphasizes the possibility that the superantigen‐reactive T cells participate in the triggering of autoimmunity in pemphigus.

Genome sequence of a clinical isolate of dermatophyte, Trichophyton rubrum from India

Trichophyton rubrum is one of the major causative agents of dermatophytosis in humans worldwide. We report the draft genome sequence of T. rubrum var. raubitschekii from Delhi, India, isolated from a patient presenting symptoms of onychomycosis. The total estimated genome size of the clinical isolate is 25.2 MB containing 8265 predicted protein-coding sequences, 91 tRNA and 15 rRNA genes. Sequence analysis of the secreted subtilases, one of the major virulence factors in dermatophytes, clusters them into three subfamilies with distinct sequence features. The genome sequence is a step in understanding diversity of dermatophytes worldwide and will aid in identification of virulence factors and dissecting mechanisms of pathogenesis among them.

Tumor necrosis factor (TNF)-α −308G/A (rs1800629) polymorphism distribution in North India and its association with pemphigus: Case–control study and meta-analysis

Abstract Pemphigus is an autoimmune blistering disorder of skin and/or mucosal surfaces characterized by intraepithelial lesions and immunoglobulin-G autoantibodies against desmogleins (proteins critical in cell-to-cell adhesion). Genetic, immunological, hormonal, and environmental factors are known to contribute to its etiology. Tumor necrosis factor-alpha (TNF-α) which plays a key role in pathogenesis of many infectious and inflammatory diseases has been found in high levels in lesional skin and sera of pemphigus patients. However, studies on association of single nucleotide polymorphism (SNP) in promoter region of TNF-α at position −308 affecting G to A transition with pemphigus has been scarce. This study was conducted to evaluate the TNF-α −308G/A SNP distribution in North Indian cohort, and to define the association between the TNF-α −308G/A SNP distribution and pemphigus, globally, by means of meta-analysis. TNF-α −308G/A SNP in pemphigus patients was investigated by cytokine genotyping using genomic DNA by PCR with sequence-specific primers. Meta-analysis of the data, including four previously published studies from other populations, was performed to generate a meaningful relationship. The results of our case–control study indicate non-significant differences between patients and controls in TNF-α −308G/A SNP. The meta-analysis also revealed that TNF-α −308G/A SNP is not associated with pemphigus risk in population at large; however, it may be contributing towards autoimmune phenomenon in pemphigus by being a part of its multi-factorial etiology. This study provides evidence that the TNF-α −308G/A polymorphism is not associated with overall pemphigus susceptibility. Nevertheless, further studies on specific ethnicity and pemphigus variants are necessary to validate the findings.

CYP2D6*4 polymorphism, tramadol treatment and its clinical impact in patients with postherpetic neuralgia

AIMnThe aim of this study was to investigate the associations between the CYP2D6*4 polymorphism, interindividual differences in CYP2D6 activity and adverse drug effects in postherpetic neuralgia (PHN) patients receiving tramadol.nnnPATIENTS & METHODSnThe study comprised 158 patients (including 78 nonresponders and 80 responders) with PHN who were undergoing analgesic treatment at the Pain Clinic in the Out Patient Department of the University College of Medical Sciences, Guru Teg Bahadur Hospital (New Delhi, India). The numerical rating scale scores were measured at the resting and movement stages; Neuropathic Pain Symptom Inventory scores were evaluated by the treating physician. WHO-brief questionnaire scores for quality of life and adverse drug effects during the time of study were recorded. All samples were analyzed for the CYP2D6*4 polymorphism using the PCR-restriction fragmentation length polymorphism method.nnnRESULTSnThe genotype distribution did not vary significantly among different age groups in nonresponders and responders. The CYP2D6*4 polymorphism was significantly associated with lower Neuropathic Pain Symptom Inventory (burning, squeezing stabbing and pressure) scores. The quality-of-life (sociological, psychological and environmental domains) scores correlated with CYP2D6*4 and showed significant results (p < 0.05) using a generalized linear model. No association was found between the physiological domain compared with the CYP2D6*4 allele (p > 0.05). In addition, the homozygous mutated CYP2D6*4 allele was not related to adverse effects of analgesic therapy.nnnCONCLUSIONnThe CYP2D6*4 polymorphism may not be a predictor for treatment outcome of patients with PHN receiving tramadol. However, further investigation is required to confirm these findings in a larger sample size.

Scrotal Swellings Synopsis of Differential Diagnosis (Part III)

Scrotal swellings enshrine clinical conditions affecting dermis and /or sub-cutaneous tissue, and those of scrotal contents. A clear cut insight into their symptoms and signs are imperative to comprehend before arriving at their precise diagnosis. A concerted endeavor in this direction has been made to appraise the audience with their nomenclature origin, clinical variants, presentation and clear cut diagnostic criteria. Accordingly angioedema, lichen simplex chronicus, orchitis, epididymitis related epididymo-orchitis, varicocele, testicular torsion, and hydrocele formed the subject matter. The role of Ultrasonography, Ultrasonic colour doppler imaging and magnetic resonance imaging in evaluating scrotal swellings has succinctly been emphasized to enrich the core curriculum of the viewers. *Corresponding author: Virendra N. Sehgal, MD, Dermato-Venerology (Skin/VD) Center, Sehgal Nursing Home, A/6 Panchwati, Delhi-110 033, India, Tel: 9810182241; E-mail: drsehgal@ndf.vsnl.net.in / sehgalvn@yahoo.co.in Citation: Sehgal, V.N., et al. Scrotal Swellings Synopsis of Differential Diagnosis (Part III). (2016) Invest Dermatol Venereol Res 2(2): 84-90. Scrotal Swellings: Synopsis of Differential Diagnosis (Part III) Virendra N Sehgal1*, Ruchi Sehgal1, Deepa Sehgal1, Shyam S Pandey2, Syed S Amin3, Sambit N Bhattacharya4, Rakesh Oberai5 Received date: May 6, 2016 Accepted date: August 7, 2016 Published date: October 15, 2016 DOI: 10.15436/2381-0858.16.905 Invest Dermatol Venereol Res | Volume 2: Issue 2 Sehgal, V.N., et al. and exit/ slipping of small bowel into the scrotum, the inguinal hernia are its other presentations. It is, therefore, worthwhile to take stock of these conditions by their salient clinical presentation, diagnosis criteria including investigation like Ultrasonography[19], Ultrasonic colour Doppler imaging[20] and magnetic resonance imaging (MRI)[21,22] to form the perspective clinical diagnosis. Accordingly, their cardinal briefs are defined in the adjoining tables (table 1 to 4) for at a glance evaluation. Scrotal swellings of dermis and sub-cutaneous tissue Angioedema (Figure. 1a), lichen simplex chronicus (Figure. 2a & b): Nomenclature, clinical variants, salient presentation and diagnostic criteria are outlined in Table 1. Figure 1a: Scrotal swellings: Acquired angioedema affecting the scrotum. Figure 2a,b: Scrotal swellings: Depicting lichen simplex chronicus. Table 1 : Scrotal Swellings: Angioedema and Lichen simplex chronicus (LSC). Nomenclature Clinical variants Presentation Diagnostic criteria Angioedema[6], angiooedema, /Quincke’sedema,/ angioneurotic edem Acquired angioedema (AAE)[7] Recurrent transitory swelling affecting the face, lips, tongue, limbs, and genitals, meditation by angiotensin-converting-enzyme inhibitor (ACE inhibitor) Hereditary angioedema(HAE)[8] Caused by a genetic mutation inherited in an autosomal dominant form, distinguished by the underlying genetic abnormality. In addition to the clinical picture supplement of haemogram, serum, electrolytes, liver and kidney functions are mandatory Clinical as well as laboratory test, inhibitor of complement enzyme, C1 esterase (C1INH) for HAE[23] Rapid swelling, edema of the dermis, subcutaneous tissue, mucosa and sub mucosal tissues. Lichen simplex chronicus (LSC) [9,24] Lichen amyloidosis[10,25,26] Intense, intermittent pruritus, scrotal erythema and edema Erectile dysfunction(ED)[27] Stable pruritic plaque(s), marked by thickening, pigmentation exaggerated skin markings, the lichenification complemented by histopathology. Thickening of the skin, variable scaling arising secondary to repetitive scratching / rubbing. The re-crossed keratinocytes that forms keratinocytes-derived amyloid in the dermis. Scrotal swellings of the contents of the scrotum Orchitis, Epididymitis and Epididymo-orchitis (Figure 3) The origin of nomenclature, clinical variants, presentation and diagnostic criteria are succinctly defined in the adjoining (table 2) www.ommegaonline.org Scrotal Swellings Invest Dermatol Venereol Res | Volume 2: Issue 2 85 Figure 3: Scrotal swellings: Epididymo-orchitis. Table 2: Scrotal Swellings: Orchitis, Epididymitis and Epididymo-orchitis. Nomenclature Clinical variants Presentation Diagnostic criteria Orchitis [10,11,28] Vide infra painful urination/ ejaculation, Swollen and tender scrotum, Blood stained semen or abnormal discharge, Enlarged prostate, Enlarged groin lymph nodes and fever. Inferno[29] Brown novel) colour Doppler ultrasound, a sign of orchitis. Inflammation of the testicles caused either by bacteria or virus, affecting one or both Epididymitis[11,12,28,30] Discomfort or pain[31] Acute, Chronic Chills, low-grade fever, Pain/discomfort in the pelvic area, painful, red, warn and swollen scrotum, pain and tenderness in the testicles, enlarged groin lymph nodes, painful, frequent urination or bowel movements, painful intercourse and ejaculation. Clinical Vide-infra see below Epididymo-orchitis [14,15,32,33] Acute gonococcal[34] / non-gonococcal urethritis (NGU)[35-40]/ non specific urethritis (NSU), Chronic Rapid swelling of epididymis and testis in a short course, a day or so. Painful, enlarged, tender and red scrotum. Gram Stain of Urethral is charge with Intracellular Neisseria, gram-negative diplococcai Chlamydia trachomatis[41] Testing Sensitivity in Midstream urine specimens Inflammation of the epididymis and/or testis Varicocele and testicular torsion: The origin of nomenclature, clinical variants, presentation, and diagnostic criteria are shown for at a glance diagnosis in (table 3) Table 3: Scrotal Swellings; Varicocele; Testicular torsion Diagnosis Clinical variants Presentation Diagnostic criteria varicocele[16,28,42] enlargement of pampiniform plexus resulting from engorgement / swollen veins apparent as a testicular swelling, variable age group being 13 30 years. Classified into Grade 1: Smallest size Grade 2: Size between Grade 1 and 2 Grade 3: Largest size Idiopathic /Pri-mary varicocele Secondary varicocele Visible/palpable enlarged veins Dragging/ aching scrotal pain/ heaviness Atrophy of the testicle(s) Benign prostatic hyperplasia (BPH) Infertility[43,44] Changing levels of testosterone ColorDoppler ultrasound (CDU)[46] presence of a venous plexus and the sum of the diameters of veins in the plexus change of flow on Valsalva maneuver / manoeuvre (Fig. 4, fig. 5) Testicular torsion:[17,47-49] Twisting of the spermatic cord resulting in ischemia of the testicles, Congenital Sudden, severe, testicular pain and tenderness in the groin and lower abdomen Clinical color ultrasound scan of the scrotum, eliciting absence of blood flow in the twisted testicle[50] Hydrocele Testis and Inguinal Hernia: There nomenclature’s origin clinical variants, presentation, and diagnostic criteria are shown in table 4, Fig 4, 5 (Figure 6a & b) 86 Sehgal, V.N., et al. Scrotal Swellings Invest Dermatol Venereol Res | Volume 2: Issue 2 www.ommegaonline.org Scrotal Swellings Invest Dermatol Venereol Res | Volume 2: Issue 2 87 Figure 4: Bilateral testis showing normal flow. Figure 5: Color Doppler image showing multiple veins dilated and filling up on valselva maneuver. The pampiniform vein measures 4 mm. Figure 6a,b: Scrotal swellings: Hydrocele depicting scrotal swelling. Table 4: Scrotal Swellings: Hydrocele Testis and Inguinal Hernia. Diagnosis Clinical variants[53] Presentation Diagnostic criteria Hydrocele Testis[51-53] Hydrocele testis a painless swelling of the scrotum due to circumscribed collection of fluid in the tunica vaginalis testis Primary hydrocele Soft, non-tender, large swelling, non palpable testis Trans-illumination positive Ultrasound mandatory for testicular visualization Secondary hydrocele due to disease(s) of testis. Acute/chronic epididymo-orchitis,Torsion of testis Testicular tumor Hematocele Filarial hydrocele Post herniorrhaphy Hydrocele of an hernial sac Fluctualating, non-reducible swelling No cough impulse It is more of a symptom than an actual pathological entity Palpation of testis impossible except for funicular and encysted hydrocele Trans-illumination elicitable Diffusion-weighted MRI of the testis[54] Apparent diffusion co-efficient (ADC) Reduced ADC values of testis, indicating reduced diffusion of the testis, with an increasing amount of fluid.(Figure 7a & b) Infantile hydrocele Infertility(?)[55] Congenital hydroceles Encysted hydrocele of the cord Inguinal hernia Groin hernia Herniation of small bowel through the external inguinal ring to scrotum. Apparent, reducible scrotal swelling Pain/discomfort while coughing, exercise, or bowel movements. Magnetic resonance imaging (MRI)[57] a diagnostic tool of high predictive value An exit/slipping of a bowel from the abdomen through the wall of the cavity into the scrotum[51,56]. It may either be direct or indirect hernias, which may or may not extend down to the scrotum. Femoral hernia Herniation of part of the abdominal contents, passing through weak area, the femoral ring at the posterior wall of the femoral canal. These swellings do not extend to the scrotum but rather are found below and lateral to the superficial inguinal ring Worsens in the day comfortable while lying. severe pain on strangulation 88 Sehgal, V.N., et al. Scrotal Swellings Invest Dermatol Venereol Res | Volume 2: Issue 2 Figure 7a & b: Scrotal swellings: Axial T2 Weighted images showing mild fluid in left scrotal sac (white arrowleft testis, white arrowhead-