Dermoscopy as an efficient aid to diagnose pigmentary and vascular component of periorbital melanosis: A cross-sectional study.

INTRODUCTION: Periorbital melanosis (POM) has a significant impact on quality of life. There is a dearth of literature regarding POM, especially in Asian population. Accurate classification of POM is contributory to the choice of therapy. The aim of this study was to assess the role of dermoscopy in etiologic classification of POM. METHODS: Two hundred and fifty adult patients (age > 18 years) of POM were enrolled over a period of 2 years. POM was classified clinically and re-evaluated based on their dermoscopic findings (pattern of pigmentary and vascular involvement, surface changes). RESULTS: Pigmented, vascular, structural, and mixed types of POM represented 6.4%, 4.8%, 0.4%, and 88.4%, respectively. Dermoscopy was found to be statistically more accurate in classifying pigmentary and vascular types of POM as compared to clinical examination with the naked eyes (p- value <0.05). Patchy or post-inflammatory pigmentation was the most common pigmentary pattern on dermoscopy (53.8%) followed by pseudoreticular (37.1%) and mixed pattern (9.1%). 80.4% patients had vascular involvement of which the most common vascular pattern was telangiectasia (58.8%). Wrinkling or increased skin laxity as a cause of shadow effect was found in 52% patients. Diffuse erythema and scaling suggestive of subtle inflammation was detected in 47.6% patients on dermoscopy. CONCLUSION: Dermoscopy as a diagnostic modality is more accurate in detecting pigmentary and vascular causes of POM and subtle signs of active inflammation in the form of erythema and scaling which is difficult to detect clinically. The major limitations of this study were lack of correlation of dermoscopic findings with the histological findings and the absence of any control group for comparison.

Grade 2 disability among leprosy patients: A pilot study from an endemic area of Central India.

Background and Aim: Deformities and disabilities in leprosy lead to significant morbidity to the individual and financial burden to the family. As the prevalence of leprosy is higher in central India, this study was conducted to know the burden of deformity and disability among leprosy patients in central India and the factors associated with it. Materials and methods: This was a hospital-based, cross-sectional study, conducted on 50 new or on treatment or released from treatment leprosy patients, attending the Dermatology OPD of a tertiary care institute in Central India. Disability was graded as per the WHO criteria for disability grading of hands and feet. Results: Grade 2 disability was found among 32% of the patients with leprosy, whereas a total of 60% of our patients had either grade 1 or grade 2 disabilities. Trophic ulcer was found among 20% and claw hand among 14% of leprosy patients. We found significant associations of lepromatous leprosy with trophic ulcer (p = 0.004) and grade 2 disability (p = 0.012). All patients with claw hands and 80% of the patients with trophic ulcers presented to the healthcare facility at least 6 months after the onset of symptoms. Conclusions: We found very high rates of disability and deformity among leprosy patients indicating possible huge burden of morbidity among leprosy patients in central India. The significant association of lepromatous leprosy with trophic ulcer and grade 2 disability indicated delayed presentation and diagnosis of lepromatous leprosy patients. Our results underlined the need for large scale community-based studies on disability and deformity among leprosy patients in Central India. These results also call for better training of primary level healthcare workers for early detection and management of deformities and disabilities as well as better patient education for leprosy patients regarding hand and foot care.

Phenotypic and genotypic characterization of biofilm forming, antimicrobial resistant, pathogenic Escherichia coli isolated from Indian dairy and meat products.

Escherichia coli are commensal gastrointestinal microflora of humans, but few strains may cause food-borne diseases. Present study aimed to identify antimicrobial resistant (AMR), biofilm-forming E. coli from Indian dairy and meat products. A total of 32 E. coli isolates were identified and evaluated for biofilm-formation. EMC17, an E. coli isolate was established as a powerful biofilm-former that attained maximum biofilm-formation within 96 h on glass and stainless-steel surfaces. Presence and expression of virulence-associated genes (adhesins, invasins and polysaccharides) and ability to adhere and invade human liver carcinoma HepG2 cell lines implicates EMC17 to be pathotype belonging to Extra-intestinal Pathogenic E. coli (ExPEC). Antibiotic profiling of EMC17 identified it as multi-drug resistant (MDR) strain, possessing extended spectrum ß-lactamases (ESBL's) and biofilm phenotype. Early production of quorum sensing molecules (AHLs) alongside EPS production facilitated early onset of biofilm formation by EMC17. Furthermore, the biofilm-forming genes of EMC17 were significantly upregulated 3-27 folds in the biofilm-state. This study showed prevalence of MDR, biofilm-forming, pathogenic E. coli in Indian dairy and meat products that potentially serve as reservoirs for transmission of antimicrobial-resistant (AMR) genes of bacteria from food to humans and pose serious food safety threat.

In vitro and in situ abrogation of biofilm formation in E. coli by vitamin C through ROS generation, disruption of quorum sensing and exopolysaccharide production.

Emergence of antimicrobial drug-resistance amongst food-borne pathogens has led to severe deficit of available therapeutics and requires novel interventions. This study determined the activity of vitamin C (VitC), a natural antioxidant as powerful antibacterial agent against multidrug-resistant (MDR), biofilm-forming E. coli. Our findings revealed that VitC wield antibacterial action in dose-time dependent manner with minimum inhibitory concentration (MIC) of 125 mM. At these concentrations VitC impaired quorum sensing (QS) and exopolysaccharide (EPS) production and induced sugar and protein leakage from the bacterial cells by virtue of reactive oxygen species (ROS) generation. Furthermore, VitC-treated bacteria showed downregulation of genes underpinning biofilm signaling (luxS) and regulation (bssR) by up to 27-folds. Finally, this study demonstrated the promising antimicrobial application of VitC, in situ, in Indian soft cheese (paneer) when applied as a coating. Therefore, VitC can be applied as natural and safe 'antimicrobial' against biofilm-forming bacteria in food systems vis-à-vis other conventional antimicrobials.

An instance of excellent response of subcutaneous zygomycosis to itraconazole monotherapy.

Subcutaneous zygomycosis is a rare fungal infection caused by Basidiobolus ranarum. This entity is usually endemic in South India and only limited numbers of cases have been reported from central India. We report a case of 4-year-old male child from Chhattisgarh, who presented with a painless, non-tender, large, subcutaneous swelling of 4 months duration on the right upper arm, which was initially misdiagnosed as benign spindle cell tumor and advised surgical resection. But the fungal culture of the affected tissue grown Basidiobolus ranarum and the child responded very well to itraconazole therapy only.

Zinc-Supported Multiwalled Carbon Nanotube Nanocomposite: A Synergism to Micronutrient Release and a Smart Distributor To Promote the Growth of Onion Seeds in Arid Conditions.

In the current scenario, nanotechnological applications in the agriculture sector showing potential impacts on the improvement of plant growth in terms of protection and safety are at a very nascent stage. The present study deals with the synergistic role of zinc (Zn) and multiwalled carbon nanotubes (MWCNTs) synthesized as a zinc oxide (ZnO)/MWCNT nanocomposite, a prospective applicant to modulate the micronutrient supply and enhance the growth of onion seeds, thereby replacing harmful, unsafe chemical fertilizers. To the best of our knowledge, this is the first report wherein MWCNTs have been envisaged as a micronutrient distributor and a nutrient stabilizer enhancing the growth of onion plant under arid conditions. The growth trend of onion seeds was evaluated in an aqueous medium with varied concentrations of (i) MWCNTs, (ii) zinc oxide nanoparticles, and (iii) ZnO/MWCNT nanocomposites. ZnO/MWCNT nanocomposites with 15 µg/mL concentration displayed the best seedling growth with the maximum number of cells in telophase. A significant growth trend with increased concentration of ZnO/MWCNTs displayed no negative impact on plant growth in contrast to that with the use of MWCNTs. The synergistic impact of Zn nanoparticles and MWCNTs in ZnO/MWCNT nanocomposites on the rate of germination was explained via a mechanism supported by scanning transmission electron microscopy.

Multi-centric validation of an in-house-developed beacon-based PCR diagnostic assay kit for Chlamydia and Neisseria and portable fluorescence detector.

OBJECTIVE: The development of an accurate, sensitive, specific, rapid, reproducible, stable-at-room-temperature and cost-effective diagnostic kit, and a low-cost portable fluorescence detector to fulfil the requirements of diagnostic facilities in developing countries. METHODS: We developed the 'Chlamy and Ness CT/NG kit' based on molecular beacons for the detection of Chlamydia trachomatis (CT) and Neisseriagonorrhoeae (NG). Multi-centric evaluation of the CT/NG kit was performed using the commercially available nucleic acid amplification test (NAAT)-based FTD Urethritis basic kit for comparison from December 2014 to November 2016. The stability of the kit reagents at 4 and 37 ˚C and the inter-day reproducibility of results were also analysed. RESULTS: The sensitivity and specificity of the kit were found to be 95.83 and 100.00 % for the detection of C. trachomatis and 93.24 and 99.75 % for N. gonorrhoeae, respectively, when tested against the commercial kit. The positive predictive value (PPV) was 100.00 and 98.57 %, whereas the negative predictive value (NPV) was 99.54 and 98.79 % for C. trachomatis and N. gonorrhoeae, respectively. Analysis of the kappa statistics enhanced the 'inter-rater' κ=0.976 for Chlamydia and κ=0.943 for Neisseria. CONCLUSION: Our kit was found to be as sensitive and specific as commercially available kits. Its low cost and ease of use will make it suitable for the routine diagnosis of C. trachomatis and N. gonorrhoeae in the resource-limited settings of developing countries.

Supplementing zinc oxide nanoparticles to cryopreservation medium minimizes the freeze-thaw-induced damage to spermatozoa.

The sperm DNA integrity post cryopreservation of human semen samples is one of the serious concerns in human infertility treatment. In the present study, the beneficial effects of zinc oxide nanoparticles in preserving the functional ability of spermatozoa was explored. Ejaculates of normozoospermic men cryopreserved along with Zinc oxide nanoparticles (ZnONPs) exhibited non-significantly higher percentage of total and progressive motility in frozen-thawed samples compared to control. The sperm chromatin damage and malondialdehyde (MDA) level was significantly lower in ZnONPs group (P < 0.01 and P < 0.05 respectively) and the spermatozoa's ability to undergo acrosome reaction was also unaltered. Fluorescence microscopy and High resolution transmission electron microscopy analysis demonstrated that the ZnONPs do not penetrate the membrane of spermatozoa but stay around the spermatozoa. In conclusion, the presence of ZnONPs during cryopreservation appears to be beneficial to the spermatozoa as they withstand freeze-thaw process competently better than control, without any adverse effect shown.

The Chronic Stages of Bovine Fasciola hepatica Are Dominated by CD4 T-Cell Exhaustion.

Fasciola hepatica infection of ruminants leads to non-resolving chronic infection, as patency develops, there is switching to a TGF-ß and IL-10 led response. Here, we explore the responses of CD4 T-cells within the major draining lymph nodes. We found minimal expression of Foxp3 within CD4 cells but elevated levels within the γδ (WC1+) population. There is a strong T-cell-intrinsic exhaustion phenotype within the hepatic lymph node (HLN) characterized by a lack of antigen-specific proliferation and cytokine secretion. CD4 T-cells recovered from the HLN had high levels of PD-1 expression and low levels of IL-2 secretion. Exogenous IL-2 partially rescued this defect; when combined with neutralization of IL-10 and TGF-ß, full restoration of proliferation, and cytokine production was achieved. Moreover, there is a clear uncoupling of the mechanisms that facilitate this regulation with parasite-specific proliferation and cytokine secretion being governed by independent means. These data would suggest that there is a CD4 T-cell-intrinsic regulation in place early in chronic infection, potentially leading to failure in resistance to reinfection.

Mutation pattern in the genome of Neisseria gonorrhoeae and its association with multidrug-resistant isolates from Delhi, India.

Mutations in PenA, PorB, MtrE and MtrR genes responsible for antimicrobial resistance were checked in 27 drug-resistant clinical isolates of Neisseria gonorrhoeae (NG). Phenotype PIB (88.88%) and mutation at G120 and A121 positions of porB were recurrent. N122K, a novel mutation, was observed in PorB in three resistant isolates. Substitution H105Y in MtrR was widespread (37% of clinical isolates). The presence of a novel mutation (L33V), along with G45D mutation in MtrR, was associated with less-resistant isolates, in contrast to isolates with G45D mutation alone. African-type penicillinase-producing NG plasmid was observed most frequently (17/27) in penicillin-resistant isolates.

A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management.

Diagnosis of Neisseria gonorrhoeae using molecular beacon.

Neisseria gonorrhoeae is an important sexually transmitted diseases (STD) causing pathogen worldwide. Due to absence of an affordable diagnostic assay, routine screening of gonococcal infection becomes impossible in developing countries where infection rates are maximum. Treatment is given on the basis of symptoms alone which leads to spread of infection. Thus, development of a rapid, sensitive, specific, and PCR based visual diagnostic assay suitable for developing countries, required for better disease management, is aimed at in present study. Endocervical swabs were collected from patients visiting gynecology department of various hospitals in Delhi. In-house PCR based assay was developed and modified to visual assay using molecular beacon for end-point detection. It was evaluated against Roche AMPLICOR NG kit and rmp gene. Specificity of beacon was confirmed by competition experiments. Diagnostic test was 98.21% specific and 99.59% sensitive whereas negative and positive predicted value were 99.40% and 98.78%, respectively. We also observed that twice the concentration (2X) of premix was stable at 4°C for 4 months and dry swab samples gave concordant results with that of wet swabs. These features make the test best suitable for routine diagnosis of genital infections in developing countries.

Seroprevalence of antibodies against Pkn1, a novel potential immunogen, in Chlamydia trachomatis-infected Macaca nemestrina and human patients.

Chlamydia trachomatis (CT) is an important cause of sexually transmitted genital tract infections (STIs) and trachoma. Despite major research into chlamydial pathogenesis and host immune responses, immunoprotection has been hampered by the incomplete understanding of protective immunity in the genital tract. Characterized vaccine candidates have shown variable efficacy ranging from no protection to partial protection in vivo. It is therefore a research priority to identify novel chlamydial antigens that may elicit protective immune responses against CT infection. In the present study we assessed the seroprevalence of antibodies against protein kinase1 (Pkn1), DNA ligaseA (LigA), and major outer membrane protein A (OmpA) following natural CT infection in humans and in experimentally induced CT infection in Macaca nemestrina. Antigenic stretches of Pkn1, LigA, and OmpA were identified using bioinformatic tools. Pkn1, LigA, and OmpA genes were cloned in bacterial expression vector and purified by affinity chromatography. Our results demonstrate significantly high seroprevalence of antibodies against purified Pkn1 and OmpA in sera obtained from the macaque animal model and human patients infected with CT. In contrast no significant seroreactivity was observed for LigA. The seroprevalence of antibodies against Pkn1 suggest that nonsurface chlamydial proteins could also be important for developing vaccines for C. trachomatis.

Cellular Neurothekeoma in a Female with Guillain-Barré Syndrome: A Case Report and Review of the Literature.

Cellular neurothekeoma is a rare cutaneous tumor that occurs more frequently in women. A 68-year-old female with a history of left nasal alar basal cell carcinoma and Guillain-Barré syndrome presented to the clinic with a 3-mm firm skin-colored papule with scattered telangiectasias. Histopathologic examination with immunochemistry of the lesion was consistent with cellular neurothekeoma. It stained positive for microphthalmia transcription factor and NKI-C3 and negative for HMB-45 and S-100. The lesion was excised with 3-mm margins, and no recurrence was noted within 1 year of follow-up. We present a case of cellular neurothekeoma in a patient with a history of Guillain-Barré syndrome as well as a review of the literature. Our case report is unique in that no prior association has been found in the literature between cellular neurothekeoma and Guillain-Barré syndrome.

An insight into the drug resistance profile & mechanism of drug resistance in Neisseria gonorrhoeae.

Among the aetiological agents of treatable sexually transmitted diseases (STDs), Neissseria gonorrhoeae is considered to be most important because of emerging antibiotic resistant strains that compromise the effectiveness of treatment of the disease - gonorrhoea. In most of the developing countries, treatment of gonorrhoea relies mainly on syndromic management rather than the aetiological based therapy. Gonococcal infections are usually treated with single-dose therapy with an agent found to cure > 95 per cent of cases. Unfortunately during the last few decades, N. gonorrhoeae has developed resistance not only to less expensive antimicrobials such as sulphonamides, penicillin and tetracyclines but also to fluoroquinolones. The resistance trend of N. gonorrhoeae towards these antimicrobials can be categorised into pre-quinolone, quinolone and post-quinolone era. Among the antimicrobials available so far, only the third-generation cephalosporins could be safely recommended as first-line therapy for gonorrhoea globally. However, resistance to oral third-generation cephalosporins has also started emerging in some countries. Therefore, it has become imperative to initiate sustained national and international efforts to reduce infection and misuse of antibiotics so as to prevent further emergence and spread of antimicrobial resistance. It is necessary not only to monitor drug resistance and optimise treatment regimens, but also to gain insight into how gonococcus develops drug resistance. Knowledge of mechanism of resistance would help us to devise methods to prevent the occurrence of drug resistance against existing and new drugs. Such studies could also help in finding out new drug targets in N. gonorrhoeae and also a possibility of identification of new drugs for treating gonorrhoea.

Prevalence of Chlamydia infection among women visiting a gynaecology outpatient department: evaluation of an in-house PCR assay for detection of Chlamydia trachomatis.

BACKGROUND: Screening women for Chlamydia trachomatis infection in developing countries is highly desirable because of asymptomatic infection. The existing diagnostic methods in developing countries are not effective and their sensitivity fall below 45.0% which leads to further spread of infection. There is an urgent need for improved and cost effective diagnostic tests that will reduce the burden of sexually transmitted infections in the developing world. METHODS: Prevalence of C. trachomatis infection among women visiting gynaecology department of Hindu Rao hospital in Delhi, India was determined using Roche Amplicor Multi Well Plate kit (MWP) as well as using in-house PCR assay. We used 593 endocervical swabs for clinical evaluation of the in-house developed assay against Direct Fluorescence Assay (DFA; Group I n = 274) and Roche Amplicor MWP kit (Group II, n = 319 samples) and determined the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of the in-house developed assay. RESULTS: We detected 23.0% positive cases and there was a higher representation of women aged 18-33 in this group. An in-house PCR assay was developed and evaluated by targeting unique sequence within the gyrA gene of C. trachomatis. Specificity of the reaction was confirmed by using genomic DNA of human and other STI related microorganisms as template. Assay is highly sensitive and can detect as low as 10 fg of C. trachomatis DNA. The resolved sensitivity of in-house PCR was 94.5% compared with 88.0% of DFA assay. The high specificity (98.4%) and sensitivity (97.1%) of the in-house assay against Roche kit and availability of test results within 3 hours allowed for immediate treatment and reduced the risk of potential onward transmission. CONCLUSIONS: The in-house PCR method is cost effective (~ 20.0% of Roche assay) and hence could be a better alternative for routine diagnosis of genital infection by C. trachomatis to facilitate improved screening and treatment management.

Comparison of an in-house PCR assay, direct fluorescence assay and the Roche AMPLICOR Chlamydia trachomatis kit for detection of C. trachomatis.

To improve the control of Chlamydia trachomatis infection in India, a rapid, specific and cost-effective method is much needed. We developed an in-house PCR assay by targeting a unique genomic sequence encoding a protein from the C. trachomatis phospholipase D endonuclease superfamily that produces an amplified fragment of 368 bp. The specificity of the primers was confirmed using genomic DNA from other sexually transmitted disease-causing and related micro-organisms and from humans. The assay was highly sensitive and could detect as low as 10 fg C. trachomatis DNA. Clinical evaluation of the in-house-developed PCR was carried out using 450 endocervical specimens that were divided in two groups. In group I (n=274), in-house PCR was evaluated against the direct fluorescence assay. The resolved sensitivity of the in-house PCR method was 97.22 % compared with 88 % for the direct fluorescent antibody assay. In group II (n=176), the in-house PCR was compared with the commercial Roche AMPLICOR MWP CT detection kit. The resolved sensitivity of the in-house PCR assay reported here was 93.1 % and the specificity was 97.46 %, making it a cost-effective alternative for routine diagnosis of genital infection by C. trachomatis. The method should facilitate early detection leading to better prevention and treatment of genital infection in India.