A prospective observational study on species differentiation and antifungal susceptibility pattern in patients with genital candidiasis.

Background: Candidial balanitis, balanoposthitis and vulvovaginitis can be diagnosed by direct microscopy, culture and treated with antifungals. Resistance to antifungals is emerging. Hence, we conducted a study to identify the causative species and antifungal susceptibility. Aim: To observe the species differentiation and antifungal susceptibility pattern in patients with genital candidiasis. Materials and Methods: A prospective observational study was carried out that included 54 patients of age group (18-60 years) diagnosed clinically and direct microscopically (KOH) for genital candidiasis. Culture was done using Sabouraud dextrose agar. Species identification and antifungal susceptibility were tested. Descriptive data were expressed in the form of frequency and percentage. Results: Out of 54 patients, 41 had culture positive candidiasis. Among the isolated species, 68.3% were Candida albicans (28/41) and 31.7% were non- albicans Candida spp. Among non-albicans Candida species (13/41), Candida glabrata (19.5%), Candida tropicalis (7.3%), Candida guilliermondii (2.4%), Candida parapsilosis (2.4%) were identified. Antifungal susceptibility was tested for fluconazole (FLU), clotrimazole (CLTZ), itraconazole (ITZ), ketoconazole (KTZ), voriconazole (VOR), amphotericin-B (AMPH-B). Except C. glabrata and C.parapsilosis, all other species were sensitive to all tested antifungals. All isolated species were sensitive to KTZ, VOR, AMPH-B, and CLTZ. Nearly 22% of isolates were resistant to fluconazole. Conclusion: C. glabrata causes complicated, severe recurrent vulvovaginitis which is fluconazole resistant. Drug sensitivity prior prescribing antifungal agent identifies appropriate drug, decreases patient's disease morbidity and cross resistance.

Evaluation of ELISA BP180 and BP230 Autoantibodies in Blister Fluid and Serum in the Diagnosis of Bullous Pemphigoid.

Bullous pemphigoid (BP) is a chronic subepidermal immunobullous disorder. Studies have demonstrated the presence of antibasement membrane zone antibodies (BP180 & BP230) in the blister fluid using enzyme-linked immunosorbent assay (ELISA). To detect and compare BP 180 and BP 230 autoantibodies in the blister fluid and serum of patients with BP by ELISA method. A total of 30 patients diagnosed as BP and not on treatment were included in the study. Blister fluid and serum were subjected to ELISA, and the results were compared. The sensitivity of ELISA BP 180 was found to be 95.8% in the blister fluid and 88.4% in the serum. The sensitivity of ELISA BP 230 in the blister fluid and serum was 20% and 16.6%, respectively. Association between ELISA antibodies done in blister and serum was analysed using Chi-square test and found to be statistically significant with P value <0.05. Blister fluid is an effective alternative to the serum in detecting BP 180 and BP 230 antibodies, especially in uncooperative and elderly patients with poor venous access.

Utility of the pattern-based approach using BIOCHIP-indirect immunofluorescence in the evaluation of subepidermal bullous disorders.

BACKGROUND: Subepidermal bullous disorders (SEBD) are a heterogeneous group of vesiculobullous diseases because of antibody-mediated destruction of proteins of the dermo-epidermal junction. Direct immunofluorescence (DIF) is the gold standard for diagnosis. BIOCHIP-indirect immunofluorescence (IIF) is a novel serological test that combines multiple target antigens in a single field. The present study aimed to evaluate the utility of the pattern-based approach in BIOCHIP-IIF for the diagnosis of SEBD. METHODS: Seventy cases of BIOCHIP-IIF that showed clinical, histopathological, and/or DIF features favoring SEBD were included in the study. The interpretation in the BIOCHIP was categorized into one of the following patterns. Pattern I: basement membrane zone (BMZ) staining in monkey esophagus (ME), primate salt-split skin (SSS)-roof staining, BP180+ and/or BP230+; Pattern II: roof staining in SSS, BP180- and BP230- with or without BMZ staining in ME; Pattern III: floor staining in SSS, BP180- and BP230-; and pattern IV: negative in SSS and other substrates. The findings were correlated with histopathology and/or DIF. RESULTS: Fifty (71.5%) cases showed pattern I or the typical bullous pemphigoid (BP) pattern. Eight (11.4%) cases showed pattern II. Patterns III and IV were observed in seven (10%) and five (7.1%) cases, respectively. BP was the most common diagnosis in patterns I and II, while anti-p200 pemphigoid was most common in pattern III, as confirmed by immunoblotting. The sensitivity of pattern I in the diagnosis of BP was 96%. CONCLUSION: BIOCHIP-IIF showed a good correlation with DIF and histopathology in the diagnosis of SEBD. This can be used as a first-line investigation in case of bullous disorders.

9-Demethoxy-medicarpin: A potential bone health supplement for the management of protein deficiency-induced bone loss in growing rats.

Human skeleton requires an adequate supply of many different nutritional factors for optimal growth and development. The role of nutrition in bone growth has piqued interest in recent years, especially in relation to maximizing peak bone mass and reducing the risk of osteoporosis. Protein deficiency-induced bone loss was induced in female growing rats. All experimental rodent diets were prepared as per recommendations for growing animals. 9-Demethoxy-medicarpin (DMM) treatment was given to growing Sprague Dawley (SD) rats at 1 mg and 10 mg dose orally for 30 days. Bones were collected for bone mineral density (BMD). Bone marrow cells were isolated from femur for calcium nodule formation. Serum samples were collected for biochemical parameters. We found that DMM treatment speeds up the recovery of musculoskeletal weakness by replenishing nutrients in proven rodent model. DMM supplementation for four weeks showed significantly increased vertebral, femur and tibial BMD compared with the untreated PD group. Albumin levels were significantly enhanced in treatment groups, in which 10 mg dose imparted a better effect. We conclude that DMM treatment led to increased BMD and biochemical parameters in protein deficient condition in growing rats and has potential as a bone growth supplement.

Evaluation of the Role of BIOCHIP Mosaic Based Indirect Immunofluorescence and ELISA BP 180 and BP 230 Autoantibodies in the Diagnosis of Bullous Pemphigoid Patients.

Background: Bullous pemphigoid (BP) is characterized by tissue-bound and circulating Immunoglobulin G (IgG) autoantibodies against BP 180 and BP 230. Diagnosis of BP is a multi-step procedure. Enzyme-linked immunosorbent assay (ELISA) is a quantitative analysis of target antigens, whereas BIOCHIP mosaic-based indirect immunofluorescence (IIF) has a combination of screening and target antigen-specific substrates in a single miniature incubation field. This study is done to compare BIOCHIP mosaic based IIF and ELISA for the diagnosis of BP. Materials and Methods: A total of 42 biopsy and/or direct immunofluorescence (DIF) proven BP patients were included in the study. Serum was subjected to BIOCHIP mosaic-based IIF and ELISA. The results were then compared. Results: Using ELISA, the sensitivity of BP 180 and BP 230 was 92.3% and 54.5%, respectively. The sensitivity of BP 180 and BP 230 by BIOCHIP was 77.4% and 60%, respectively. The association between ELISA and BIOCHIP was analyzed using the Chi-square test and was found to be statistically significant with a P value ≤ 0.05. Conclusion: Our study concluded that both BIOCHIP and ELISA showed comparable sensitivity in diagnosing BP. Both are non-invasive, less time-consuming, and provide fast results. However, BIOCHIP can delineate bullous pemphigoid from other sub-epidermal bullous diseases.

Evaluation of the Role of Oral Mucosal Direct Immunofluorescence and Salivary Desmoglein 1 and 3 Enzyme-Linked Immunosorbent Assay in Patients With Oral Mucosal Pemphigus.

Background: Pemphigus vulgaris (PV) is characterized by antibodies against desmosomal adhesion proteins desmoglein (Dsg) 1 and 3 which can be detected by direct immunofluorescence (DIF) or enzyme-linked immunosorbent assay (ELISA). Oral lesions usually precede cutaneous lesions and an early diagnosis can prevent mortality and morbidity. Dsg antibodies can be detected by ELISA in saliva of patients with oral mucosal pemphigus. This study compares oral mucosal DIF with the salivary Dsg1 and 3 ELISA. Materials and Methods: A total of 26 biopsy and/or DIF-proven PV patients with oral erosions without cutaneous lesions were included in the study. Biopsy of oral mucosa was taken for DIF by standard method. Saliva sample was obtained and processed for ELISA. The results were then compared. Results: Out of 26 patients, 22 (84.6%) had a positive oral mucosal DIF and four patients (15.4%) had negative DIF. Nine patients (34%) had positive salivary Dsg3 ELISA. Seven patients (27%) had positive salivary Dsg1 ELISA. Taking oral DIF as the gold standard, the sensitivity of salivary Dsg1 ELISA was 31.8% and of salivary Dsg3 ELISA was 40.9%. Conclusion: Although DIF is the gold standard for the diagnosis of PV, salivary Dsg1 and 3 ELISA can also be used in the diagnosis of oral pemphigus.

Cladrin alleviates dexamethasone-induced apoptosis of osteoblasts and promotes bone formation through autophagy induction via AMPK/mTOR signaling.

Glucocorticoid-induced osteoporosis (GIOP) is a common clinical consequence that arises due to the extensive usage of glucocorticoids. Cladrin (Clad), a methoxylated isoflavone has been reported to have a bone protecting effect by enhancing osteoblast proliferation and differentiation. However, its consequences on GIOP are not reported yet. This study investigates whether Clad protects against the deleterious effects of Dexamethasone (Dex) on osteoblast and bone. Mice calvarial osteoblasts were treated with Clad and then exposed to Dex to study the effect on osteoblast differentiation, proliferation, and survival. Further, GIOP mice were treated with Clad (5 and 10 mg/kg) doses along with reference standard alendronate (ALN 3 mg/kg) for evaluation of bone protecting effect of Clad. We analyzed bone and vertebral microarchitecture, mechanical strength, and biochemical parameters. We observed that Clad at 10 nM concentration mitigated Dex-induced cytotoxicity and defend osteoblasts against apoptosis. Subsequent results demonstrate that Clad suppressed apoptosis of osteoblast in the presence of Dex by enhancing autophagy in a way that was reliant on the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway. Furthermore, micro-CT scanning, eco MRI results, and serum CTX levels revealed that 12 weeks of Clad treatment prevented bone loss and preserved trabecular bone mass in GIOP animals. We also observed that Clad treated osteoblasts had a lower rate of apoptosis and a greater LC3-II/LC3-I ratio than the Dex group. Our findings show that Clad can protect osteoblasts against glucocorticoids by inducing autophagy via the AMPK/mTOR pathway.

Discontinuation of PTH therapy amplifies bone loss by increasing oxidative stress: An event ameliorated by sequential IL-17 neutralizing antibody therapy.

Osteoporosis leads to excessive bone resorption which is not accompanied by equal amount of bone formation. PTH (1-34) forms the mainstay of bone anabolic therapy. Intermittent PTH (iPTH) has the ability to reconstruct skeleton, a property not shared by other anti-resorptives. In initial phases of PTH treatment, bone formation exceeds bone resorption. However, gradually this phase is replaced by increased bone resorption. Thus, a replacement post PTH discontinuation is much needed. Studies with bisphosphonates and Denosumab post PTH withdrawal have yielded promising but variable results. Thus, there is scope for trying new combinations. Our previous studies have shown the superior skeletal effects of neutralizing IL17 antibody (NIL17) over anti-RANKL antibody. Thus, here we investigated if sequential treatment of NIL17 after PTH withdrawal (SHIFT) could serve as a promising therapeutic approach for osteoporosis treatment. Our results show that PTH withdrawal (PTH-W) led to mitigation of its anabolic effects as evidenced by reduced BMD, bone trabecular and cortical microarchitectural parameters. In the continuous PTH (PTH-C) and the Shift group, all these parameters were preserved as par with the sham group. Shift therapy also significantly increased PINP levels. Most importantly, serum CTX-I levels and osteoclast numbers, which were elevated in PTH groups were significantly suppressed in NIL17 monotherapy and shift group. Also, expression of FOXO1 and ATF-4, the main regulators of redox balance and function in osteoblasts, were found to be enhanced maximally in the sequential therapy group. Our study thus advocates use of NIL17 as a replacement therapeutic option post PTH discontinuation.

Unusual Presentation of Trichoblastoma.

We report a 39-year-old man who presented with skin-colored plaque over the glabella and root of the nose. Histopathology revealed the diagnosis of trichoblastoma. This case is reported to emphasize the rare presentation of trichoblastoma as it usually presents as an isolated nodule.

Quality Improvement Initiative to Improve Hand Hygiene Compliance in Indian Special Newborn Care Unit.

Pilot data on Hand hygiene (HH) compliance using a standard World Health Organisation checklist for 1-week suggested only 20% compliance. So, we planned a Quality Improvement study to improve HH compliance among health care providers in our Special Newborn Care Unit from 20% to 60% over 12 months. METHODS: We did this study in 3 phases: Baseline phase (2 months), Intervention phase (8 months), and Postintervention phase (2 months). A multidisciplinary Quality Improvement team composed of doctors, nursing staff, and ward attendants was constituted. The team analyzed potential barriers to HH by Fishbone analysis. Three trained observers randomly selected two target Special Newborn Care Unit patients daily and collected data on HH compliance unobtrusively during the three 8-h shifts over 24 h. In addition, we tested a range of interventions using multiple Plan Do Study Act cycles: Staff education; Displaying posters; Round the clock availability of soap and hand rub; Staff felicitation; Group performance feedback. We also collected data on healthcare-associated infections in all three phases. RESULTS: The total observations for HH during the baseline, intervention, and postintervention phase were 1488, 5808, and 1464, respectively. The HH compliance improved from 27.2% to 57.1% in the postintervention phase. There was no difference in the healthcare-associated infections among the three phases. CONCLUSIONS: The HH compliance rates improved significantly but not to the desired extent. So, we planned to increase our workforce, and improve our training program and infrastructure.

Extract and fraction of Musa paradisiaca flower have osteogenic effect and prevent ovariectomy induced osteopenia.

BACKGROUND: Osteoporosis is an asymptomatic bone disorder leading to altered bone microarchitecture, mineralization and strength. Musa paradisiaca has been reported to have antioxidant and anti-inflammatory effects in various diseases. Its impact on postmenopausal osteoporosis has not been investigated yet. PURPOSE: The intention of the current study was to evaluate the bone regeneration and osteoprotective potential of extract and fraction of M. paradisiaca flower in ovariectomized (Ovx) Sprague Dawley (SD) rats, a model of post-menopausal bone loss. The study also aims to identify osteogenic compounds from active fraction. METHODS: Ethanolic extract (MFE) and butanolic fraction (MFE-Bu) from flower of M. paradisiaca were prepared and their efficacy was tested in rat femur osteotomy model at different doses. Effective dose from both extract (250 mg/kg) and fraction (50 mg/kg) were taken for study in osteopenic bone loss model. PTH was taken as reference standard (20 µg/kg/twice a week). Bones were harvested at autopsy for dynamic and static histomorphometry. Serum was collected for ELISA. Pure compounds were isolated from butanolic fraction (MFE-Bu), and were assessed for their osteogenic effect. RESULTS: MFE and MFE-Bu were observed for their potential in bone healing and prevention of bone loss. Both MFE and MFE-Bu promoted new bone regeneration at injury site as assessed by microCT and calcein dye labeling studies. These also led to restoration of bone microarchitecture deteriorated as a result of osteopenia and improved bone biomechanical properties. Extract as well as the fraction exhibited dual bone anabolic and anti-resorptive properties where they elevated serum procollagen type I N-terminal propeptide (P1NP), a bone formation marker and suppressed serum C-telopeptide of type I collagen (CTX-1), a bone resorption marker. As many as four osteogenic compounds were isolated from MFE-Bu. Oleracein-E was found to be the most potent osteogenic agent based on osteoblast differentiation, mineralization assays, qPCR and protein expression studies. CONCLUSION: Our studies demonstrates that ethanolic extract from the flower of M. paradisiaca and its butanolic fraction exhibit dual osteogenic and anti-resorptive potential, and have an advantage over PTH which though promotes bone formation but is also bone catabolic in nature.

Anti-P 200 pemphigoid - The most common floor binding subepidermal autoimmune bullous disease in a tertiary care center in south India.

BACKGROUND: The pemphigoid group of diseases may present clinically and immunologically in a very similar fashion. Indirect immunofluorescence microscopy with readily available salt-split human skin in a BIOCHIP™ helps to classify these conditions as those with either with roof binding or floor binding of immunoreactants. Epidermolysis bullosa acquisita, anti-laminin 332 pemphigoid and anti-p200 pemphigoid show floor binding, while in the most frequent type of pemphigoid disease, bullous pemphigoid, epidermal side staining pattern is seen on salt-split skin Aims: The aim of the study was to detect the target antigens in sub-epidermal bullous diseases. METHODS: Forty patients with bullous pemphigoid diagnosed by lesional histopathology and direct immunofluorescence microscopy were re-evaluated by a BIOCHIP™ mosaic containing both tissue substrates and recombinant target antigens. Sera with floor pattern staining on salt-split skin were further evaluated by immunoblotting with dermal extract. RESULTS: Five patients with floor staining had anti-p200 pemphigoid. LIMITATIONS: We could not perform serration pattern analysis of direct immunofluorescence in our patients. CONCLUSION: Histopathology and direct immunofluorescence microscopy cannot differentiate between various entities of pemphigoid diseases. A multivariant approach using a BIOCHIP™ mosaic including salt-split skin followed by immunoblotting with dermal extract helps to identify the target antigen.

Comparative Analysis of BIOCHIP Mosaic-based Indirect Immunofluorescence with Direct Immunofluorescence in Diagnosis of Autoimmune Bullous Diseases: A Cross-Sectional Study.

BACKGROUND: Autoimmune bullous diseases (AIBD) are a heterogeneous group of diseases characterized by autoantibodies against desmosomal proteins in the pemphigus group of disorders and adhesion molecules of the dermal-epidermal junction in pemphigoid group of diseases. Direct immunofluorescence (DIF) establishes the diagnosis of AIBD by demonstrating intercellular deposits of IgG and C3 in case of pemphigus and linear deposits of IgG and C3 along the basement membrane zone (BMZ) in bullous pemphigoid (BP). BIOCHIP mosaic-based indirect immunofluorescence (IIF), a novel diagnostic approach employs detection of characteristic staining pattern and target antigens in a single miniature incubation field. AIM: To compare the BIOCHIP mosaic-based IIF with DIF in the diagnosis of AIBD. MATERIALS AND METHODS: A total of 40 patients of AIBD in the active phase of the disease were included in the study. Skin biopsy was done in these patients for DIF study and serum was subjected to BIOCHIP mosaic-based IIF assay. The results were then compared. RESULTS: DIF revealed a diagnosis of Pemphigus in 18 patients and BP in 22 patients. BIOCHIP showed a diagnosis of pemphigus in 18 patients, BP in 18 patients and floor pattern staining in four patients, which could be attributed to any of the floor pattern staining subepidermal blistering disease. LIMITATIONS: Small sample size, lack of control group and no comparison made with ELISA. CONCLUSION: This study concludes that the result of BIOCHIP shows correlation with the DIF and can be used as a first line-screening tool in the diagnosis of AIBD.