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Neonatal dengue is an under-diagnosed disease likely due to low index of suspicion along with its resemblance to sepsis. We hereby report two cases of neonatal dengue, highlighting the need of high degree of suspicion in infants born to febrile mothers even with maternal serology being negative. Moreover, severity of neonatal illness positively correlates with the maternal disease.

Most of the mutations occurred in SARS-CoV-2 are either relatively neutral or swiftly purged. However, some mutations have altered the functional aspects in terms of infectivity and transmission, host-viral interactions, disease severity and immune or vaccine escape. There are emerging evidence that certain mutations are jeopardizing the immune based therapies. The present research report is focused on the identification of genomic signatures of SARS-CoV-2 variant that caused mortality during second and third wave of COVID-19 in Western Rajasthan, India. We identified that Delta clade of SARS-CoV-2 is the predominant cause of mortality during second wave and even third wave in Western Rajasthan, India. Importantly, this study also revealed the unique and common substitution mutations within the spike domain, those are present in mortality and survived persons during the second and third wave of COVID-19 in India. In addition, this study also revealed the multiple lineage markers (Delta and Omicron), that would update with insightful understanding in the clade development of SARS-CoV-2.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objective To determine the species distribution of causative agents causing dermatophytosis, and their antifungal susceptibility pattern for terbinafine and itraconazole in trichophyton spp among samples collected in patients with dermatophytosis clinical suspicion during the period of December 1, 2020 to January 31, 2022. Materials and Methods This is a prospective study conducted in the Department of Microbiology of a tertiary care super specialty and referral Centre of western Rajasthan from December 1, 2020 to January 31, 2022. Skin scraping, nail clipping, and hair pluckings were collected in mycology lab from clinically suspected cases of dermatophytosis presenting to the department of dermatology for conventional identification, and antifungal susceptibility testing. The specimens were subjected to direct KOH and calcofluor white microscopy and conventional fungal culture on SDA at 25 ͦ C and 37 ͦ C. The cultures positive for dermatophytes were speciated by microslide culture lactophenol cotton blue mount, hair perforation test, and urease test. The isolates identified as Trichophyton spp were taken up for antifungal susceptibility testing against terbinafine and itraconazole by microbroth dilution according to CLSI-M38 A2 guidelines. Further terbinafine resistance gene evaluation for detection of C1191A and T1189C single nucleotide polymorphism in Squalene epoxide by Amplified Refractory Mutation System-Polymerase chain Reaction (ARMS-PCR) is undergoing for trichophyton spp. Results Over the 14-month study period, the laboratory processed total of 174 specimens: 134 skin scraping, 36 nail clipping, and 4 hair pluckings. Of them, 106 (61.62%) specimens were microscopy positive and 111 (63.79%) were culture positive. Out of the 111 culture-positive agents isolated, 94 (84.68%) were found to be dermatophytes. On isolate profiling of 94 dermatophytes T. mentagrophyte was found to be most common 45 (48%) followed by T. rubrum 27 (29%), T. tonsurans 20 (21%), T. verrucosum 1 (1.1%), and Microsporum spp 1(1.1%). Antifungal susceptibility of 93 Trichophyton spp against terbinafine showed resistance among 58.06% isolates with 83.33% isolates among terbinafine resistant cases showing ≥4 μg/ml minimum inhibitory concentration. There was no resistance detected for itraconazole with microbroth dilution. Conclusion A total of (54.02%) skin, hair, nail infections were found to be caused by dermatophytes. On isolate profiling, T. mentagrophyte, T. rubrum, and T. tonsurans were found to be predominant species among our isolates showing altered trend of local isolates from T. tonsurans being second most common spp isolated in past. On antifungal susceptibility >55% isolates showed resistance for Terbinafine with >80% having higher MIC of ≥4 μg/ml on the contrary there was no observed resistance for itraconazole. There is a need for encouraging dermatologists for prescribing routine fungal microscopy, culture, and AFST for dermatophytes in Western Rajasthan, to reduce the indiscriminate use of antifungals.

Background SARS-CoV-2 is the causative agent of worldwide pandemic disease coronavirus disease 19. SARS-CoV-2 bears positive sense RNA genome that has organized and complex pattern of replication/transcription process including the generation of subgenomic RNAs. Transcription regulatory sequences have important role in the pausing of replication/transcription and generation of subgenomic RNAs. Results In the present bioinformatics analysis, a consensus secondary structure was identified among negative sense subgenomic RNAs of SARS-CoV-2. This consensus region is present at the adjacent of initiation codon. Conclusions This study proposed that consensus structured domain could involve in mediating the long pausing of replication/transcription complex and responsible for subgenomic RNA production.

BackgroundTraditional medicine systems such as Ayurveda contain a vast repository of naturally occurring herbs with strong antimicrobial potency. A multitude of complementary therapies have been explored for their therapeutic role to treat COVID-19 during the pandemic. There have been promising results reported from in silico, in vitro and in vivo studies that need to be further explored in humans.ObjectiveThe present randomized placebo control trial evaluates the clinical efficacy of an integrated approach including Ayurvedic polyherbal formulation, NOQ19 in the improvement of mild to moderate category of COVID-19 infected patients.Patients and methodsA multicentric, randomized, placebo control design was adopted for the study. A total of 76 patients with positive COVID-19 RT-PCR test were enrolled in the study; 39 in the NOQ19 arm and 37 in the placebo arm. Patients were randomized and blinded to their respective intervention, which was provided along with the standard of care treatment. Rate of recovery assessment was done on Day 3, 5 and 7 via RT-PCR test to measure the rate of recovery. Blood markers were analysed on Day 0 and Day 7.OutcomesThe patients were assessed for rate of recovery via RT-PCR and improvement in blood biomarkers. They were also monitored for any adverse events or side effects of the drug.ResultsThe present study demonstrated a significant early recovery in the patients who took the NOQ19 formulation. Patients who received NOQ19 with the standard care of treatment showed clinical improvement in terms of oxygen requirement, breathlessness and SpO2, though the difference was not statistically significant. Moreover, no side effects were observed with the use of NOQ19.ConclusionAn overall integrated approach of standard of care treatment with Ayurvedic formulation (NOQ19) results in early clinical outcome in the management of mild to moderate cases of COVID-19.

The neurological manifestation following a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is varied, and till now, only a few studies have reported the same.We used retrospective data from May to July 2021 and prospective study data from August to September 2021, including that from children aged between one month and 18 years who presented to a tertiary care referral center with the neurological manifestation and had a history of coronavirus disease 2019 (COVID-19) infection or exposure and positive SARS-CoV-2 serology. The neuroradiological manifestations were further categorized as in a predesigned proforma.Case records of the 18 children who fulfilled the criteria were included in the study; among them, seven (38.8%) were male and 11 (61.1%) were female. Predominant presentation in our study group was status epilepticus (six of 18) and Guillain-Barré syndrome (five of 18). Other manifestations included stroke (two of 18), demyelinating syndromes (three of 18), and autoimmune encephalitis (two of 18). Most of the children had favorable outcomes except for one mortality in our cohort.Delayed complications following SARS-CoV-2 infection are seen in children. A temporal correlation was noted between the COVID-19 infection and the increasing number of neurological cases after the second wave. Steroids could be beneficial while treating such patients, especially in the presence of high inflammatory markers. Testing for SARS-CoV-2 serology during the pandemic can give a clue to the underlying etiology. Further multicentric studies are required to understand the varied neurological manifestations following SARS-CoV-2 infection in children.

During October 2020, Delta variant was detected for the first time in India and rampantly spread across the globe. It also led to second wave of pandemic in India which affected millions of people. However, there is limited information pertaining to the SARS-CoV-2 strain infecting the children in India.Here, we assessed the SARS-CoV-2 lineages circulating in the pediatric population of India during the second wave of the pandemic. Clinical and demographic details linked with the nasopharyngeal/oropharyngeal swabs (NPS/OPS) collected from SARS-CoV-2 cases (n = 583) aged 0-18 year and tested positive by real-time RT-PCR were retrieved from March to June 2021.Symptoms were reported among 37.2% of patients and 14.8% reported to be hospitalized. The E gene CT value had significant statistical difference at the point of sample collection when compared to that observed in the sequencing laboratory. Out of these 512 sequences 372 were VOCs, 51 were VOIs. Most common lineages observed were Delta, followed by Kappa, Alpha and B.1.36, seen in 65.82%, 9.96%, 6.83% and 4.68%, respectively in the study population.Overall, it was observed that Delta strain was the leading cause of SARS-CoV-2 infection in Indian children during the second wave of the pandemic. We emphasize on the need of continuous genomic surveillance in SARS-CoV-2 infection even amongst children.

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1–4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy., Graphical Abstract, Developing a successful vaccine against dengue has been challenging. Arun Sankaradoss and colleagues developed a genotype-specific DNA DENV vaccine candidate that elicits robust dengue immune responses. The low cost and thermostability of DNA vaccines should allow countries to implement large-scale vaccination programs, which could greatly reduce dengue cases.

The pandemic of coronavirus disease has created a global emergency after its origin in China, in December 2019. We investigated SARS-CoV-2 infection in the local population of a prime city of Western Rajasthan in India and found that the co-morbidities like diabetes (54.3%), cardiovascular diseases (28.8%), chronic respiratory conditions (18.4%) are the predominant risk factors for acquiring the infection. The young adult male population from urban area were maximum affected by the disease. Most common presenting complains were dry cough (65.7%), fever (62.3%) followed by sore throat (24.1%). A declining positive case percentage was found over the study period, though the number of samples received for SARS-CoV-2 testing increased with time, implying the effectiveness of government policies at community level to spread the outbreak.

Brucellosis is a significant bacterial zoonotic disease with a high seroprevalence in low-to-middle-income countries where there is a significant contact of humans with animals. This prospective study was conducted to observe the seroprevalence of brucellosis in 75 symptomatic patients with pyrexia of unknown origin (PUO) and 75 high-risk individuals (10 veterinarians, 15 milkmen and 50 healthy contacts of symptomatic patients) with possible exposure to brucellosis. Serum samples collected from these patients and individuals were subjected to rose Bengal test (RBT) and ELISA for the detection of IgM and IgG antibodies. RBT was positive in 50 samples (40 PUO and 10 high risk), of which 25 (33.3%) PUO cases had detectable IgM antibodies, whereas IgG antibodies were detected in 20 patients. None of samples from high-risk individuals was reactive for IgM/IgG antibodies by ELISA. The overall seroprevalence in this study was 16.7% (33.3% in PUO patients). Being a predominantly livestock rearing area, brucellosis is a significant health-care problem in this part of India with this entity being linked to 33% of PUO cases.

Following the recent clinical clearance of an Indian DNA COVID-19 vaccine, India and Africa are potential regions where DNA vaccines may become a major delivery system subject to a range of immunological and regulatory scrutiny. The ongoing COVID pandemic highlights the need to tackle viral variants and expand the number of antigens and assess diverse delivery systems. To address some of these key issues, we have created a Dengue DNA vaccine candidate with the EDIII region as the key antigen given the promise of this segment in not causing ADE, a challenge with this disease. In addition, we have added the NS1 region to broaden the immune response. Following a large Dengue viral sequencing exercise in India, complemented with data from east Africa, our approach was to generate a consensus of four serotypes ED3-NS1 vaccine to explore tackling the issue of diversity. OurIn silicostructural analysis of EDIII consensus vaccine sequence revealed that epitopes are structurally conserved and immunogenic across HLA diversity. Vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Furthermore, the DNA vaccination confers protection against DENV challenge in AG129 mice. Finally, assaying of intracellular staining for IFN-γ, immunoglobulin IgG2(a/c) /IgG1 ratios as well as immune gene profiling suggested a strong Th1-dominant immune response. Our Dengue DNA platform with a focus on Indo-African sequences offers an approach for assessing cross reactive immunity in animal models and lays the foundation for human vaccine roll out either as a stand-alone or mix and match strategy.

Background: India introduced BBV152/Covaxin and AZD1222/Covishield vaccines from January 2021. We estimated effectiveness of these vaccines against severe Coronavirus disease 2019 (COVID-19) among individuals aged >=45 years. Methods: We did a multi-centric, hospital-based, case–control study between May and July 2021. Cases were severe COVID-19 patients and controls were COVID-19 negative individuals from 11 hospitals. Vaccine effectiveness (VE) was estimated for full (2 doses ≥14days) and partial (1 dose ≥21 days) vaccination; duration between two vaccine doses and against the Delta variant. We used a random effects logistic regression model to calculate adjusted odds ratios (aOR) with 95% CI after adjusting for relevant known confounders. Findings: We enrolled 1,143 cases and 2,541 controls. The VE of full vaccination was 80% (95% CI: 73%-86%) with AZD1222/Covishield and 69% (95% CI: 54%-79%) with BBV152/Covaxin. The VE was highest for a gap of 6-8 weeks between two doses of AZD1222/Covishield (92%, 95% CI: 82%-96%) and BBV152/Covaxin (92%, 95% CI: 26%-99%). The VE estimates were similar against the Delta strain and sub-lineages. Interpretation: BBV152/Covaxin and AZD1222/Covishield were effective against severe COVID-19 among the Indian population during the period of dominance of highly transmissible Delta variant in second wave of pandemic. An escalation of two-dose coverage with COVID-19 vaccines is critical to control the pandemic in the country. Funding Information: Indian Council of Medical Research Declaration of Interests: None to declare. Ethics Approval Statement: We obtained written informed consent from all the participants or their legally authorized representatives. Study procedures were approved by the Institutional Human Ethics Committees of all participating institutions.

We studied the pattern and duration of viral ribonucleic acid (RNA) shedding in 32 asymptomatic and 11 paucisymptomatic coronavirus disease 2019 cases. Viral RNA shedding in exhaled breath progressively diminished and became negative after 6 days of a positive reverse-transcription polymerase chain reaction test. Therefore, the duration of isolation can be minimized to 6 days.

Dengue virus (DENV) infection, estimated to cause infection approximately 390 million people each year globally of which 96 million develop clinical disease. Dengue serotype 2 (DEN-2) is the most prevalent serotype over the past 50 years in India but serotype 3 and 4 have appeared in some epidemics as well. A retrospective study was conducted in a teaching hospital, western India, between January 2014 and December 2018. The records of dengue serological test were analyzed. A total 40 randomly selected non-structural protein 1 (NS1) antigen positive samples were analyzed by reverse transcription-polymerase chain reaction. The demographic data, i.e., age and sex, along with geographic location and platelet count level was recorded from Serology laboratory register and Hospital Information System. A total 14.85% (735/4948) samples were tested positive for dengue serology. Most of the laboratory confirmed dengue cases 34.97% (257/735) were seen in 21-30 years of age group. Most common serotype detected in the tested samples was DEN-3 in 55% (22/40, 13 mono-infection and 9 co-infection with DEN-1 and DEN-2) cases. Present study gives an insight about the trend of dengue seropositivity among suspected cases in western part of Rajasthan, India. This study showed a higher seroprevalence of dengue infection as well as a gradual increase in the seroprevalence in this part of India. This article is protected by copyright. All rights reserved.

Background Government of India is committed to eliminate measles and control rubella/congenital rubella syndrome (CRS) by 2020. In 2016, CRS surveillance was established in five sentinel sites. We analyzed surveillance data to describe the epidemiology of CRS in India. Methodology/Principal findings We used case definitions adapted from the WHO-recommended standards for CRS surveillance. Suspected patients underwent complete clinical examination including cardiovascular system, ophthalmic examination and assessment for hearing impairment. Sera were tested for presence of IgM and IgG antibodies against rubella. Of the 645 suspected CRS patients enrolled during two years, 137 (21.2%) were classified as laboratory confirmed CRS and 8 (1.2%) as congenital rubella infection. The median age of laboratory confirmed CRS infants was 3 months. Common clinical features among laboratory confirmed CRS patients included structural heart defects in 108 (78.8%), one or more eye signs (cataract, glaucoma, pigmentary retinopathy) in 82 (59.9%) and hearing impairment in 51. (38.6%) Thirty-three (24.1%) laboratory confirmed CRS patients died over a period of 2 years. Surveillance met the quality indicators in terms of adequacy of investigation, adequacy of sample collection for serological diagnosis as well as virological confirmation. Conclusions/Significance About one fifth suspected CRS patients were laboratory confirmed, indicating significance of rubella as a persistent public health problem in India. Continued surveillance will generate data to monitor the progress made by the rubella control program in the country., Author summary Rubella infection during the first trimester of pregnancy can affect the fetus, resulting in spontaneous abortion, stillbirth or an infant born with a combination of birth defects known as congenital rubella syndrome (CRS). Government of India is committed to eliminate measles and control rubella/CRS and has completed nationwide immunization campaigns using measles-rubella vaccine targeting children aged 9 months to 14 years. A case-based surveillance for CRS is one of the strategies for achieving elimination. The Indian Council of Medical Research and the Ministry of Health and Family Welfare initiated surveillance for CRS in five sentinel sites to estimate the disease burden. During 2016–18, the surveillance sites enrolled 645 suspected CRS patients, 137 (21.2%) were classified as laboratory confirmed CRS and 8 (1.2%) as congenital rubella infection. Common clinical features among laboratory confirmed CRS patients included structural heart defects (78.8%), one or more eye signs (cataract, glaucoma, pigmentary retinopathy (59.9%)) and hearing impairment (38.6%). Thirty-three (24.1%) laboratory confirmed CRS patients died over a period of 2 years. The surveillance data indicated significance of rubella as persistent public health problem in India.

Malaria is a sterning public health concern in India and contribute to a major part of malaria burden in Southeast Asia. Being more populated and diverse geographic conditions makes more suitable place for sustaining malaria parasite in India. Anti-malarial resistance is a major concern in the battle against malaria, and the identified molecular markers will aid us to monitor the drug resistance in endemic areas. The aim of the current study is to determine the genotype of drug resistance associated genes pvmdr-1 and pvcrt-o from four different regions of India. Especially from Puducherry and Jodhpur, there were no prior studies focused on screening of drug resistance genes in P. vivax parasite. A total of 240 positive P. vivax infected patient samples were collected from four tertiary care hospitals from four different regions of India, namely, Puducherry (PDY), Mangaluru (MAQ), Cuttack (CTC), Jodhpur (JDH). All samples were screened by microscopy, RDT, QBC, and further DNA was extracted and vivax mono-infection was confirmed by nested PCR. Randomly selected amplicons were further subjected to nucleotide sequencing. The prevalence of K10 insertion in pvcrt-o gene was detected with 18.8% in PDY, 12.5% in MAQ and 6.3% in CTC P. vivax isolates, whereas no change in nucleotide was identified in P. vivax isolates collected from JDH region. Based on the F1076L mutation in pvmdr-1 gene, resistant P. vivax isolates was highly predominant in both the regions, JDH and CTC, with 100%, followed by MAQ with 93.3% and PDY with 73.3%. This study showed less frequency of pvcrt-o and high frequency of pvmdr-1 gene variants associated with CQ resistance, which act as an indicator and the onset of P. vivax drug resistance trend in four different regions of India. Due to the poor phenotypic studies available for P. vivax parasite, the present study data for CQ resistance based on pvcrt-o and pvmdr-1 markers should assist by providing base-line data for future monitoring of drug resistance.

Objective— To investigate the role of Pyk2, a proline-rich nonreceptor tyrosine kinase, in G protein−coupled receptor agonist, thrombin-induced human aortic smooth muscle cell growth and migration, and injury-induced vascular wall remodeling. Methods and Results— Thrombin, a G protein–coupled receptor agonist, activated Pyk2 in a time-dependent manner and inhibition of its stimulation attenuated thrombin-induced human aortic smooth muscle cell migration and proliferation. Thrombin also activated Grb2-associated binder protein 1, p115 Rho guanine nucleotide exchange factor, Rac1, RhoA, and p21-activated kinase 1 (Pak1) and interference with stimulation of these molecules attenuated thrombin-induced human aortic smooth muscle cell migration and proliferation. In addition, adenovirus-mediated expression of dominant negative Pyk2 inhibited thrombin-induced Grb2-associated binder protein 1, p115 rho guanine nucleotide exchange factor, Rac1, RhoA and Pak1 stimulation. Balloon injury also caused activation of Pyk2, Grb2-associated binder protein 1, p115 rho guanine nucleotide exchange factor, Rac1, RhoA, and Pak1 in the carotid artery of rat, and these responses were sensitive to inhibition by the dominant negative Pyk2. Furthermore, inhibition of Pyk2 activation resulted in reduced recruitment of smooth muscle cells onto the luminal surface and their proliferation in the intimal region leading to suppression of neointima formation. Conclusion— Together, these results demonstrate for the first time that Pyk2 plays a crucial role in G protein–coupled receptor agonist thrombin-induced human aortic smooth muscle cell growth and migration, as well as balloon injury–induced neointima formation.

Background: Malaria is one of the major communicable diseases in India and worldwide. PvMSP3β is a highly polymorphic gene due to its large insertions and deletions in the central alanine-rich region, which, in turn, makes it a valuable marker for population genetic analysis. Very few studies are available from India about the genetic diversity of Plasmodium vivax based on PvMSP3β gene, and hence, this study was designed to understand the molecular diversity of the P. vivax malaria parasite. The accumulating epidemiological data provide insights into the circulating genetic variants of P. vivax in India, and ultimately benefits the vaccine development. Materials and Methods: A total of 268 samples confirmed to be positive by microscopy, rapid diagnostic test, and quantitative buffy coat test were collected from four different regions of India (Puducherry, Mangaluru, Jodhpur, and Cuttack) in the present study. Polymerase chain reaction (PCR)-based diagnosis was carried out to confirm the P. vivax monoinfection, and only the mono-infected samples were subjected to PvMSP3β gene amplification and further restriction fragment length polymorphism (RFLP) to determine suballeles. Results: Based on the size of the amplified fragment, the PvMSP3β gene was apportioned into two major types, namely Type A genotype (1.6–2 Kb) was predominantly present in 148 isolates and Type B (1–1.5 Kb) was observed in 110 isolates. The percentage of mixed infections by PCR was 3.73%. All the PCR products were subjected to RFLP to categorize into suballeles and we detected 39 suballeles (A1–A39) in Type A, and 23 suballeles (B1–B23) in Type B genotype. A high degree of diversity was observed among the isolates collected from Mangaluru region when compared to isolates collected from other regions. Conclusion: The present study showed a high degree of genetic diversity of PvMSP3β gene among the isolates collected from various parts of India. High polymorphism in PvMSP3β gene makes it a promising marker for epidemiological and vaccine development studies.

Alpha-melanocyte stimulating hormone (alpha-MSH) is an endogenous anti-inflammatory peptide reported to possess antimicrobial properties, however their role as antibacterial peptides is yet to be established. In the present study, we examined in vitro antibacterial activity of alpha-MSH against S. aureus strain ISP479C and several methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) S. aureus strains. Antibacterial activity was examined by varying several parameters, viz., bacterial cell densities, growth phase, pH, salt concentration, and temperature. Antibacterial activity was also examined in complex biomatrices of rat whole blood, plasma and serum as well as in biofilm form of bacteria. Our results showed that alpha-MSH possessed significant and rapid antibacterial activity against all the studied strains including MRSA (84% strains were killed on exposure to 12 microM of alpha-MSH for 2h). pH change from 7.4 to 4 increased alpha-MSH staphylocidal activity against ISP479C by 21%. Antibacterial activity of alpha-MSH was dependent on bacterial cell density and independent of growth phase. Moreover, antimicrobial activity was retained when alpha-MSH was placed into whole blood, plasma, and serum. Most importantly, alpha-MSH exhibited antibacterial activity against staphylococcal biofilms. Multiple membrane permeabilization assays suggested that membrane damage was, at least in part, a major mechanism of staphylocidal activity of alpha-MSH. Collectively the above findings suggest that alpha-MSH could be a promising candidate of a novel class of antimicrobial agents.

OBJECTIVE—To determine the microbiological profile and antibiotic susceptibility patterns of organisms isolated from diabetic foot ulcers. Also, to assess potential risk factors for infection of ulcers with multidrug-resistant organisms (MDROs) and the outcome of these infections. RESEARCH DESIGN AND METHODS—Pus samples for bacterial culture were collected from 80 patients admitted with diabetic foot infections. All patients had ulcers with Wagner’s grade 3–5. Fifty patients (62.5%) had coexisting osteomyelitis. Gram-negative bacilli were tested for extended spectrum β-lactamase (ESBL) production by double disc diffusion method. Staphylococcal isolates were tested for susceptibility to oxacillin by screen agar method, disc diffusion, and mec A–based PCR. Potential risk factors for MDRO-positive samples were explored. RESULTS—Gram-negative aerobes were most frequently isolated (51.4%), followed by gram-positive aerobes and anaerobes (33.3 and 15.3%, respectively). Seventy-two percent of patients were positive for MDROs. ESBL production and methicillin resistance was noted in 44.7 and 56.0% of bacterial isolates, respectively. MDRO-positive status was associated with presence of neuropathy (P = 0.03), osteomyelitis (P = 0.01), and ulcer size >4 cm2 (P < 0.001) but not with patient characteristics, ulcer type and duration, or duration of hospital stay. MDRO-infected patients had poor glycemic control (P = 0.01) and had to be surgically treated more often (P < 0.01). CONCLUSIONS—Infection with MDROs is common in diabetic foot ulcers and is associated with inadequate glycemic control and increased requirement for surgical treatment. There is a need for continuous surveillance of resistant bacteria to provide the basis for empirical therapy and reduce the risk of complications.

Two hundred Staphylococcus aureus strains collected from an Indian hospital were tested for mupirocin susceptibility using disc diffusion method and E-test. High-level and low-level mupirocin resistance was detected in 10 (5%) and 2 (1%) S. aureus strains, respectively. Pulsed-field gel electrophoresis analysis of the high-level mupirocin-resistant methicillin-resistant S. aureus isolates revealed the presence of 2 clones with the majority of strains belonging to 1 clone, suggesting clonal dissemination.

To understand the role of thrombin in inflammation, we tested its effects on migration of THP-1 cells, a human monocytic cell line. Thrombin induced THP-1 cell migration in a dose-dependent manner. Thrombin induced tyrosine phosphorylation of Pyk2, Gab1, and p115 RhoGEF, leading to Rac1- and RhoA-dependent Pak2 activation. Downstream to Pyk2, Gab1 formed a complex with p115 RhoGEF involving their pleckstrin homology domains. Furthermore, inhibition or depletion of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, or Pak2 levels substantially attenuated thrombin-induced THP-1 cell F-actin cytoskeletal remodeling and migration. Inhibition or depletion of PAR1 also blocked thrombin-induced activation of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2, resulting in diminished THP-1 cell F-actin cytoskeletal remodeling and migration. Similarly, depletion of Gα12 negated thrombin-induced Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2 activation, leading to attenuation of THP-1 cell F-actin cytoskeletal remodeling and migration. These novel observations reveal that thrombin induces monocyte/macrophage migration via PAR1-Gα12-dependent Pyk2-mediated Gab1 and p115 RhoGEF interactions, leading to Rac1- and RhoA-targeted Pak2 activation. Thus, these findings provide mechanistic evidence for the role of thrombin and its receptor PAR1 in inflammation.

Recent literature suggests that cyclin-dependent kinases (CDKs) mediate cell migration. However, the mechanisms were not known. Therefore, the objective of this study is to test whether cyclin/CDKs activate Pak1, an effector of Rac1, whose involvement in the modulation of cell migration and proliferation is well established. Monocyte chemotactic protein 1 (MCP1) induced Pak1 phosphorylation/activation in human aortic smooth muscle cells (HASMCs) in a delayed time-dependent manner. MCP1 also stimulated F-actin stress fiber formation in a delayed manner in HASMCs, as well as the migration and proliferation of these cells. Inhibition of Pak1 suppressed MCP1-induced HASMC F-actin stress fiber formation, migration, and proliferation. MCP1 induced cyclin D1 expression as well as CDK6 and CDK4 activities, and these effects were dependent on activation of NFATc1. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1-induced Pak1 phosphorylation/activation and resulted in decreased HASMC F-actin stress fiber formation, migration, and proliferation. CDK4, which appeared to be activated downstream of CDK6, formed a complex with Pak1 in response to MCP1. MCP1 also activated Rac1 in a time-dependent manner, and depletion/inhibition of its levels/activation abrogated MCP1-induced NFATc1-cyclin D1-CDK6-CDK4-Pak1 signaling and, thereby, decreased HASMC F-actin stress fiber formation, migration, and proliferation. In addition, smooth muscle-specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak1 activities, resulting in reduced neointima formation in response to injury. Thus, these observations reveal that Pak1 is a downstream effector of CDK4 and Rac1-dependent, NFATc1-mediated cyclin D1 expression and CDK6 activity mediate this effect. In addition, smooth muscle-specific deletion of NFATc1 prevented the capacity of vascular smooth muscle cells for MCP-1-induced activation of the cyclin D1-CDK6-CDK4-Pak1 signaling axis, affecting their migration and proliferation in vitro and injury-induced neointima formation in vivo.

Our previous studies revealed that alpha-melanocyte stimulating hormone (α-MSH) is strongly active against Staphylococcus aureus (S. aureus) including methicillin resistant S. aureus (MRSA). Killing due to α-MSH occurred by perturbation of the bacterial membrane. In the present study, we investigated the in vitro synergistic potential of α-MSH with five selected conventional antibiotics viz., oxacillin (OX), ciprofloxacin (CF), tetracycline (TC), gentamicin (GM) and rifampicin (RF) against a clinical MRSA strain which carried a type III staphylococcal cassette chromosome mec (SCCmec) element and belonged to the sequence type (ST) 239. The strain was found to be highly resistant to OX (minimum inhibitory concentration (MIC) = 1024 µg/ml) as well as to other selected antimicrobial agents including α-MSH. The possibility of the existence of intracellular target sites of α-MSH was evaluated by examining the DNA, RNA and protein synthesis pathways. We observed a synergistic potential of α-MSH with GM, CF and TC. Remarkably, the supplementation of α-MSH with GM, CF and TC resulted in ≥ 64-, 8- and 4-fold reductions in their minimum bactericidal concentrations (MBCs), respectively. Apart from membrane perturbation, in this study we found that α-MSH inhibited ≈ 53% and ≈ 47% DNA and protein synthesis, respectively, but not RNA synthesis. Thus, the mechanistic analogy between α-MSH and CF or GM or TC appears to be the reason for the observed synergy between them. In contrast, α-MSH did not act synergistically with RF which may be due to its inability to inhibit RNA synthesis (

Treatment of serious life threatening infections due to multi-drug resistant pathogens presents a difficult challenge due to the limited therapeutic options. Therefore, we studied the in vitro susceptibility of tigecycline, a new glycylcycline with promising broad spectrum of activity against Gram positive and Gram negative bacteria at a tertiary care hospital in north India.A total of 75 multi-drug resistant isolates of methicillin resistant Staphylococcus aureus (21), vancomycin resistant enterococci (14), vancomycin resistant Streptococcus spp. (3), extended spectrum beta lactamase producing Gram negative bacteria (11) and multi-resistant Acinetobacter spp. (26) were tested for tigecycline susceptibility by the E-test and disc diffusion methods. An additional 83 multi-resistant Gram negative clinical isolates were screened by disc diffusion method alone.All the isolates of MRSA, VRE, vancomycin resistant Streptococcus spp. and ESBL producing enteric bacteria were sensitive to tigecycline by the E-test and disc diffusion methods. However, only 42 per cent of Acinetobacter spp. were found to be sensitive to tigecycline by the E-test method.In conclusion, tigecycline was found to be highly effective against Gram-positive bacteria and Gram-negative members of Enterobacteriaceae, but a high prevalence of resistance in members of Acinetobacter spp. is worrisome.

The in vitro activity of daptomycin and selected comparator agents against Staphylococcus aureus and vancomycin-resistant Enterococcus faecium (VREF) isolates recovered from hospitalized patients with skin and soft tissue infection's was evaluated by Clinical and Laboratory Standards Institute broth microdilution method. Daptomycin was the most active agent against both S. aureus (MIC 90 , 1 μg/mL; 100% susceptible) and VREF (MIC 90 , 4 μg/mL; 100% susceptible), making it an excellent therapeutic option.

We analysed risk factors for nosocomial meticillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections (SSTIs) in three Indian hospitals. We also determined antimicrobial resistance patterns and genotypic characteristics of MRSA isolates using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal cassette chromosome (SCCmec) typing. Medical records of 709 patients admitted to three tertiary hospitals with nosocomial S. aureus SSTIs were clinically evaluated. Antimicrobial susceptibility testing of patient isolates was performed in accordance with Clinical and Laboratory Standards Institute guidelines, with meticillin and mupirocin resistance confirmed by multiplex polymerase chain reaction. PFGE analysis of 220 MRSA isolates was performed, followed by MLST and SCCmec typing of a selected number of isolates. MRSA was associated with 41%, 31% and 7.5% of infections at the three hospitals, respectively. Multiple logistic regression analysis identified longer duration of hospitalisation [odds ratio (OR): 1.78; OR: 2.83 for >or=20 days], intra-hospital transfer (OR: 1.91), non-infectious skin conditions (3.64), osteomyelitis (2.9), neurological disorders (2.22), aminoglycoside therapy (1.74) and clindamycin therapy (4.73) as independent predictors for MRSA SSTIs. MRSA isolates from all three hospitals were multidrug resistant, with fifteen clones (I-XV) recognised. A majority of the strains possessed type III cassette. The common sequence type (ST) 239 was considered the signature MLST sequence for PFGE clone III. This major MRSA clone III was closely related to the UK EMRSA-1 and was significantly more resistant to antibiotics. Dissemination of multidrug-resistant MRSA clones warrants continuous tracking of resistant genotypes in the Indian subcontinent.