Monorama Deb

Genetic characterization of Chikungunya virus from New Delhi reveal emergence of a new molecular signature in Indian isolates

BackgroundChikungunya (CHIK) is currently endemic in South and Central India and exist as co-infections with dengue in Northern India. In 2010, New Delhi witnessed an outbreak of CHIK in the months October-December. This was the first incidence of a dominant CHIK outbreak in Delhi and prompted us to characterize the Delhi virus strains. We have also investigated the evolution of CHIK spread in India.FindingsClinical samples were subjected to RT-PCR to detect CHIK viral RNA. The PCR amplified products were sequenced and the resulting sequences were genetically analyzed. Phylogenetic analysis based on partial sequences of the structural proteins E1 and E2 revealed that the viruses in the latest outbreak exhibited ECSA lineage. Two novel mutations, E1 K211E and E2 V264A were observed in all Delhi isolates. In addition, CHIKV sequences from eight states in India were analyzed along with Delhi sequences to map the genetic diversity of CHIKV within the country. Estimates of average evolutionary divergence within states showed varying divergence among the sequences both within the states and between the states. We identified distinct molecular signatures of the different genotypes of CHIKV revealing emergence of a new signature in the New Delhi clade. Statistical analyses and construction of evolutionary path of the virus within the country revealed gradual spread of one specific strain all over the country.ConclusionThis study has identified unique mutations in the E1 and E2 genes and has revealed the presence of ancestral CHIKV population with maximum diversity circulating in Maharashtra. The study has further revealed the trend of CHIK spread in India since its first report in 1963 and its subsequent reappearance in 2005.

Minimum inhibitory concentration of carbapenems and tigecycline against Salmonella spp.

Antimicrobial resistance in Salmonella spp. is of grave concern, more so in quinolone-resistant and extended-spectrum beta-lactamase (ESBL)-producing isolates that cause complicated infections. The MIC of azithromycin, ciprofloxacin, cefixime, cefepime, ceftriaxone, gatifloxacin, imipenem, levofloxacin, meropenem and ofloxacin (E-test strip) and tigecycline and faropenem (agar dilution) against 210 Salmonella spp. was determined. MIC(90) (defined as the antimicrobial concentration that inhibited growth of 90 % of the strains) of the carbapenems (imipenem and meropenem) for Salmonella Typhi and Salmonella Paratyphi A was 0.064 microg ml(-1). MIC(90) of faropenem was 0.25 microg ml(-1) for S. Typhi, S. Paratyphi A and Salmonella Typhimurium. The MIC(90) of azithromycin for all Salmonella spp. ranged from 8 to 16 microg ml(-1). Tigecycline showed an MIC(90) of 2 microg ml(-1) for S. Typhi, 1 microg ml(-1) for S. Paratyphi A and 4 microg ml(-1) for S. Typhimurium. We concluded that tigecycline and the carbapenems are likely to have roles in the final stage of treatment of quinolone-resistant and ESBL-producing multidrug-resistant salmonellae.

Clinically and Microbiologically Derived Azithromycin Susceptibility Breakpoints for Salmonella enterica Serovars Typhi and Paratyphi A

ABSTRACT Azithromycin is an effective treatment for uncomplicated infections with Salmonella enterica serovar Typhi and serovar Paratyphi A (enteric fever), but there are no clinically validated MIC and disk zone size interpretative guidelines. We studied individual patient data from three randomized controlled trials (RCTs) of antimicrobial treatment in enteric fever in Vietnam, with azithromycin used in one treatment arm, to determine the relationship between azithromycin treatment response and the azithromycin MIC of the infecting isolate. We additionally compared the azithromycin MIC and the disk susceptibility zone sizes of 1,640 S. Typhi and S. Paratyphi A clinical isolates collected from seven Asian countries. In the RCTs, 214 patients who were treated with azithromycin at a dose of 10 to 20 mg/ml for 5 to 7 days were analyzed. Treatment was successful in 195 of 214 (91%) patients, with no significant difference in response (cure rate, fever clearance time) with MICs ranging from 4 to 16 μg/ml. The proportion of Asian enteric fever isolates with an MIC of ≤16 μg/ml was 1,452/1,460 (99.5%; 95% confidence interval [CI], 98.9 to 99.7) for S. Typhi and 207/240 (86.3%; 95% CI, 81.2 to 90.3) (P < 0.001) for S. Paratyphi A. A zone size of ≥13 mm to a 5-μg azithromycin disk identified S. Typhi isolates with an MIC of ≤16 μg/ml with a sensitivity of 99.7%. An azithromycin MIC of ≤16 μg/ml or disk inhibition zone size of ≥13 mm enabled the detection of susceptible S. Typhi isolates that respond to azithromycin treatment. Further work is needed to define the response to treatment in S. Typhi isolates with an azithromycin MIC of >16 μg/ml and to determine MIC and disk breakpoints for S. Paratyphi A.

Characterization of Streptococcus pneumoniae isolates from India with special reference to their sequence types

INTRODUCTION Streptococcus pneumoniae is a major cause of mortality and morbidity in young children and the elderly. In the present study we evaluated antimicrobial susceptibilities, serotypes, and sequence types of pneumococcal isolates recovered in New Delhi, India. METHODOLOGY A total of 126 clinical isolates of Streptococcus pneumoniae were investigated. They were subjected to disk diffusion susceptibility testing, broth microdilution testing, serotyping and multilocus sequence typing. RESULTS Broth microdilution assay showed that 5%, 20% and 23% of the isolates exhibited resistance to penicillin, erythromycin and ciprofloxacin, respectively. Serotypes19, 1 and 6 were more frequently isolated. Thirty per cent of the strains were comprised of serotypes 1, 3, 5, 19A and 7F, which are not included in the seven-valent vaccine. Fifty-nine isolates were typed using multilocus sequence typing. Thirty new sequence types were encountered in this study. Only one clonal complex with 4 isolates was seen; 11 clonal complexes and 96 sequence types (STs) were observed among 115 Indian isolates. Only 18 of the 96 STs were found globally, of which only 4 STs were found in many countries with larger numbers. CONCLUSIONS This study identifies the non-vaccine serotypes of Streptococcus pneumoniae circulating in India. It is important that an appropriate vaccine which covers all serotypes is used in the region.

Prevalence of intestinal parasitic infections in HIV-infected individuals and their relationship with immune status.

BACKGROUND AND OBJECTIVES Intestinal parasitic infection is a common entity in patients infected with human immunodeficiency virus (HIV). These infections may lead to fatal complications in the immuno suppressed individuals. The aim of the present study was to determine the prevalence of intestinal parasitic infections in HIV sero-positive patients and their relationship with the immune status of individuals. MATERIALS AND METHODS Fecal samples from 100 HIV sero-positive and an equal number of HIV sero-negative individuals were collected and examined for enteric parasites by direct microscopy. CD4 counts were carried out in only HIV sero-positive patients. Prevalence of intestinal parasites in patients with CD4 count<200 cells/μl, 200-499 cells/μl, and ≥500 cells/μl in HIV-infected patients were compared. RESULTS Enteric parasites were detected in 59.3% HIV-infected patients with CD4 count<200 cells/μl as compared with 23.5% in patients with CD4 count>200 cells/μl (P<0.01). Prevalence of coccidian parasites was significantly (P<0.01) higher (14%) in HIV sero-positive subjects compared with HIV sero-negative subjects (2%). Isospora belli (25%) was the most common parasite with CD4 count<200 cells/μl, followed by Cryptosporidium parvum (12.5%). Prevalence of intestinal parasitic infections was significantly higher in patients with diarrhea, 73.6% than without diarrhea, 25.9%, (P<0.05). The mean CD4 count of HIV sero-positive patients presenting with diarrhea was significantly (P<0.01) lower (181.26±135.14) than without diarrhea (352.02±204.03). CONCLUSION This study emphasizes the need for routine screening of parasites especially in patients with lower CD4 count so as to decrease the morbidity by ensuring the early treatment of the cases.

Concomitant TB and cryptococcosis in HIV-infected patients

Four cases of concomitant tuberculosis and cryptococcosis infection in HIV-positive patients are described. As the HIV pandemic progresses and the proportion of patients with end-stage disease increases, a high suspicion of incidence and unusual forms of infections must always be kept in mind.

Characterization of non-classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges

Objective To establish the relative importance of Salmonella enterica serovar Typhi with non-classical quinolone resistance. Methods Eight hundred and ninety-one isolates of S. Typhi, isolated between 2004 and 2011, were tested for antibiotic susceptibility determination using disc diffusion and E-test. The mechanisms of fluoroquinolone resistance were studied in a sub-set of the NALS (nalidixic acid susceptible) isolates by wave nucleic acid fragment analysis of PCR products from gyrA, gyrB, parC and parE and from the plasmid borne determinants: qnrA,B,S; aac(6′)-Ib-cr and qepA. To assess genetic relatedness multi-locus variable number tandem repeat analysis was carried out using five loci. Results Eighty isolates with a nalidixic acid MIC of <32 mg/L (NALS) and a ciprofloxacin MIC of >0.064 mg/L CIPI (ciprofloxacin reduced susceptibility) were found. In 36 NALS CIPI isolates two distinct genotypes were identified when compared with 16 susceptible controls: Group B (n = 34), mutation in gyrB at codon 464, NAL MIC of 3–12 mg/L and CIP MIC of 0.064–0.5 mg/L.; and Group C, mutation in gyrA at codon 83 (n = 2) NAL MIC of 16 mg/L and CIP MIC of 0.25–0.38 mg/L. Group B isolates were found in different strain backgrounds as defined by MLVA. Conclusion The use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIPI-NALS isolates, an established phenotype in India.

In vitro antimicrobial susceptibility patterns of Propionibacterium acnes isolated from patients with acne vulgaris

INTRODUCTION Propionibacterium acnes has been implicated in the development of acne vulgaris. Rampant use of topical and systemic antibiotics for acne vulgaris has led to resistance due to selective pressure. This study aimed to determine antibiotic resistance of P. acnes. METHODOLOGY A total of 102 samples were collected from acne lesions and cultured onto sheeps blood agar and brain-heart infusion agar supplemented with 5 g/L glucose and 2 mg/L furazolidone) (BHIg) under aerobic and anaerobic conditions. Species identification was done by conventional methods and the VITEK2 Compact system. The isolates were tested for penicillin, erythromycin, clindamycin, ciprofloxacin, nadifloxacin, and tetracycline by E-test, and minimum inhibitory concentration (MIC) of minocycline was determined by agar dilution on BHIg. MIC results were interpreted as per EUCAST (European Committee on Antimicrobial Susceptibility Testing) and CLSI (Clinical Laboratory Standards Institute) guidelines. RESULTS P. acnes was the most common anaerobe (66%) isolated. Resistance rates using EUCAST and CLSI breakpoints were 10.6% and 6.1%, 7.6% and 0%, 7.8% and 0% for erythromycin, clindamycin, and minocycline, respectively. Tetracycline resistance was observed in 9.2% isolates irrespective of the interpretative criteria used. MIC50 and MIC90 values for nadifloxacin (0.25 and 1 µg/mL) were found to be twofold lower than those for ciprofloxacin (0.5 and 1 µg/mL). Similarly, MIC50 and MIC90 values for minocycline (0.125 and 0.5 µg/mL) were also two- to threefold lower than those for tetracycline (0.38 and 1 µg/mL). CONCLUSIONS To the best of our knowledge, this is the first study focusing on P. acnes resistance from India.

Detection of mutations in gyrB using denaturing high performance liquid chromatography (DHPLC) among Salmonella enterica serovar Typhi and Paratyphi A

Background Fluoroquinolone resistance is mediated by mutations in the quinolone-resistance determining region (QRDR) of the topoisomerase genes. Denaturing high performance liquid chromatography (DHPLC) was evaluated for detection of clinically important mutations in gyrB among Salmonella. Methods Salmonella Typhi and S. Paratyphi A characterised for mutation in QRDR of gyrA, parC and parE were studied for mutation in gyrB by DHPLC and validated by sequencing. Results The DHPLC analysis was able to resolve the test mutant from isolates with wild type gyrB and distinguished mutants from other mutant by peak profile and shift in retention time. Three sequence variants were detected at codon 464, and a novel mutation Ser→Thr was also detected. gyrB mutation was associated with non classical quinolone resistance (NALS-CIPDS) in 34 isolates of S. Typhi only and was distinct from classical quinolone resistance associated with gyrA mutations (NALR-CIPDS). Conclusions DHPLC is effective for the detection of mutation and can reduce the need for sequencing to detect clinically significant gyrB mutations. GenBank accession nos KF993966, KF993965 and KF993964.

Bacteraemic Haemophilus influenzae type B pneumonia complicated with empyema--report of a case and review of literature.

T 1 diabetes is caused by the immune-mediated destruction of pancreatic islet cells leading to insufficient insulin production and consequent clinical manifestations of hyperglycaemia. The etiology of type 1 diabetes, like other autoimmune diseases, can be thought of as a complex interaction between genes and the environment. It is apparent that genes cannot be acting alone. However, it is still unclear how non-genetic events may lead to disease. Environmental factors could act by either triggering an already established degree of autoimmunity, directly causing the destructive inflammatory response, or both, which then sets off a chain of events culminating in clinical diabetes. Epidemiological, histological and immunological data indicate a role for viruses in the pathogenesis of type 1 diabetes, although it has proven difficult to find a causal relationship. Current evidence strongly supports the association of the disease with infection by different enterovirus species. There is evidence that viral factors operate to influence the rate of disease progression in subjects with pancreatic autoantibodies and that persistent non-lytic enterovirus infections (not acute lytic infections) are associated with the disease. Novel methods to detect enteroviruses in samples from patients at different clinical stages of the disease (autoantibodies in the absence of hyperglycemia, clinical onset of diabetes, later clinical stages) will be presented. These include virus isolation in culture, gene amplification and sequencing, studies of tissue samples from autopsy materials and from organ donors with type 1 diabetes, specimens obtained from newly-diagnosed living patients (blood, pancreas biopsy). Identification of enterovirus types associated with diabetes in different cases and different geographic areas remains, however, highly controversial.H carcinoma (HCC) is the third leading cause of cancer mortality worldwide. Chronic infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) is the major cause of this malignancy. Recently, advanced sequencing technologies such as whole genome sequencing, exome sequencing, and RNA sequencing have provided opportunities to understand the insight of how somatic mutations, structure variations, HBV integrations and epigenetic modifications contribute to HCC development. Chronic inflammation provides “fertile field” for somatic mutation, selection, and adaptation, the so called evolutionary process during HCC development. Genomic variations of HCC caused by various etiological factors might be different, but the common genomic variations should be important to elucidate the HCC evolutionary process. Genome-wide analysis of HBV integrations may help clarifying the mechanisms of HBV-induced hepatocarcinogenesis and disease progression. RNA sequencing presents additional evidences of epigenetic modifications during HCC evolution. In this review, the current findings of next generation sequencing of HCC caused by various etiological factors to interpret the potential mechanisms of HCC evolution is summarized. Understanding the key genomic variations during HCC evolution is essential for accurate prognosis prediction and efficient targeted treatment of this fatal malignancy.A diminishing the importance of polioviruses, non-polioviruses are emerging as causative agents of severe central nervous system (CNS) involvement. Recently, outbreaks of enterovirus 71(EV71) -associated CNS infections were reported in Asia, Australia and Europe.This is the first study on genotyping of EV71 in children with meningoencephalitis in Iran. Viral RNA was extracted from 170 cerebrospinal fluid samples from children <8 years old with primary diagnosis of aseptic meningitis. Specific EV71 PCR was done to identify the genotype of the detected EV71 viruses. Human Enteroviruses (HEVs) were detected in 89 (52.3%) patients, and EV71 infection was detected in 19 (21.3%) patients out of the 89 EV positive patients, and the genotype C was identified in 15 isolates. Genotype C should be considered as the prevalent EV71 circulating genotype in Iran especially in aseptic meningitis cases.T vaccines treat a disease or condition by inducing or strengthening a pre-existing immune response. A chimeric protein comprising the core and surface regions of the hepatitis B virus (HBV) envelope protein was designed as a therapeutic vaccine. The premise was that if the HBV surface protein is fused with the core protein of the viral envelope, it can produce both cellular and humoral immune response. NNPREDICT and PSIPRED programs were used to predict the secondary structure elements of the protein. 3D-JIGSAW was used to predict the tertiary structure of the protein. The tertiary structure shows α-helices form a helical bundle domain and the β-strands form a separate domain. The provided data can be used to explain binding to antibodies found on the surface of B-cells, class II MHC molecules and T-cell receptors (TCR).A case of bacteraemic pneumonia complicated with pleural empyema due to Haemophilus influenzae type b is reported in a one-year old previously healthy child who had apparently no other associated medical condition. The organism was isolated from both the pleural fluid aspirate and blood of the patient with pneumonia. She was successfully treated with parenteral ampicillin and chloramphenicol alongwith intercostal chest tube drainage. The case is notable because it adds to the existing disease spectrum of invasive Hib diseases and brings awareness to the existing burden of the disease in Asia. In addition, it reflects the urgent need to include Hib vaccine in the current immunization program in India.R PCR has for many years been an important diagnostic tool for infectious diseases. Recently, the development of fully automated systems has generated the possibility for walk-away analyses that give rapid diagnosis, and save personnel resources. Evaluation of the performance and convenience criteria is important to find the most cost-effective solution in the routine setting. Norovirus infection can cause severe diarrhea and vomiting. It is highly contagious and requires Droplet Precautions in hospitals. Atypical pneumonia, especially Legionella pneumophila (Lpn) can cause severe pneumonia and needs specific antimicrobial treatment. Therefore, there is a growing need for rapid, sensitive methods for these pathogens. We evaluated the fully automated BD MAX rapid multiplex PCR assays developed by Diagenode: Enteric Viral Panel and Atypical Pneumonia assay. BD MAX has shorter turn-around-time and hands-on-time than our routine methods. One technician can process 24 specimens in 30 min. Results are ready after approximately 3 hours. For the Atypical Pneumonia assay, the overall concordance was 93.4% (298/319) between Atypical Pneumonia assay and our routine methods (real-time PCR based assays). Nine out of 16 Lpn positive samples (56.3%) was found positive by Atypical Pneumonia assay. The sensitivity for Lpn detection should be optimized before converting to this assay in the routine. For the Enteric Viral Panel, the overall concordance was 71.7% (33/46) between Enteric Viral Panel and our routine method (antigenic based assay). The reason for the low concordance was that the Enteric Viral Panel was more sensitive than our current method.A HIV epidemic, several countries reported co-infections of HIV and Leishmania. The co-infection of these two pathogens results into rapid disease progression, more severe disease and poor response to treatment. The first case of VL-HIV co-infection from India was published in 1999, after which several new cases of co-infections are reported. But the proportion has been low (0.029-0.4%) as in comparison to other countries where both these diseases are co-endemic, especially southern Europe. So far only 86 cases of VL-HIV and 8 cases of cutaneous leishmaniasis (CL) and HIV have been published since 1999 to date. More than 2 cases of diffuse cutaneous leishmaniasis with HIV and one case of post-kala-azar mucocutaneous leishmaniasis (PKML) have also been reported. Though the first case of VL-HIV co-infection was reported from sub-Himalayan state of Uttarakhand, most cases are reported from VL endemic region of Bihar. Similarly, most CL-HIV co-infections were published from Rajasthan, a western state of India, endemic for CL. But at least 2 cases from non-endemic states of South India are also reported. Most cases were seen during 1997-2007 and after that number of new cases has gone down. This could be due to low HIV prevalence in VL and CL endemic regions and also due to availability of free HAART for HIV infected patients. Atypical characteristics noted were high relapse and fatality rates despite treatment with liposomal amphotericin-B. Also diagnostic sensitivity of serological tests was found lower in co-infected patients as compared to HIV negative VL patients.C urine, considered a ‘miraculous’ drug used in Prophetic Medicine, since the pre-Islamic era camel milk and urine were used as drinking medicine for different health problems. In addition, camel urine was used as treatment for womens hair in the folk-medicine, and it has proven to be effective as an antimicrobial agent, and may not have side effects for humans. Furthermore, camel urine may be resistant to factors such as high temperatures and an extensive waiting period in laboratory conditions, which can reduce the effectiveness of antibiotics. The aim of our study was to examine the effectiveness of camel urine as an antifungal agent following exposure to high temperatures and long time periods in laboratory conditions. After maintaining camel urine in natural laboratory conditions for 6 weeks at temperatures of up to 100oC, we tested camel urine on the fungi Aspergillus niger and Fusarium oxysporum, and on the yeast Candida albicans. We then measured the dry weight of each microorganism, and determined their minimum inhibitory and fungicidal concentrations. Our results showed that after maintaining for 6 weeks, camel urine did not lose its antifungal activity; dry weights following treatment were decreased 100% of the dry weight prior to treatment for Aspergillus niger and Candida albicans, and 53.33% for Fusarium oxysporum. Our study demonstrates that camel urine is a highly effective and resilient antifungal agent for treating human and plant fungal diseases.T emergence of multidrug-resistant bacteria is a world health problem. In Brazil, according to data obtained from the first five years of the SENTRY Antimicrobial Surveillance Program, Methicillin-resistant S. aureus (MRSA) strains were among the most prevalent pathogens and contributed to 56% of the nosocomial and community infections. Our research group has studied the antibacterial effect of silver and polymeric nanoparticles against multi-resistant bacteria, including MRSA strains from several diseases (hospital infections and bovine mastitis). Silver nanoparticles were obtained by Fusarium oxysporum (biological silver). Silver nanoparticles combined with others antimicrobials such as phenazine (natural compound) showed synergic effect against MRSA. Biocompatible polymeric particles comprised by alginate/chitosan or chitosan/sodium tripolyphosphate (TPP) were prepared and used for the encapsulation of mercapto succinic acid (MSA) and nitrosation for sustained and controlled NO (nitric oxid)-releasing. The antibacterial activity of NO-releasing particles showed a decrease in the number of bacteria isolated from bovine mastitis. Ours studies indicate that silver and polymeric nanoparticles are an interesting approach to combat bacteria resistance and they may be a good alternative treatment to control infections caused by multi-resistant bacteria.1 Staphylococcus aureus isolates were collected from microbiology lab hospitals in Riyadh and Jeddah. Distribution of methicillin resistant Staphylococcus aureus (MRSA) isolates among clinical departments and isolations from different specimens showed that the wound infections resemble the highest percentage of the clinical sites (26%). The all over percentage of MRSA topical infections (70%) was higher than MSSA infections.All isolates were sensitive to vancomycin which is the drug of choice for MRSA infections. Mupirocin showed low degree of resistance, which was 6%. Susceptibility to other commonly used antibiotics was variable. Resistance oxacillin was the highest 34%. Some degrees of resistance to sulphomethoxazole/ trimethoprim and fusidic acid (34%) were noticed. Terms like boarder line resistant, low-level mutation, and boarderline susceptible had also been obtained in our studied isolates of S. aureus for methecillin using Minimum Inhibitory Concentration (MIC) test; which was observed in the percentage of MRSA isolates in our data that had MICs of 256μg/m1 (30.83%). Studying the distribution of phage type patterns among Staphylococcus patients isolates in Saudi cohort; it was found that only 30.56% were typable with different degree, and 69.7% were non typable. Differentiation increased in the phage typing, and genome typing yielding 20 and 23 different S. aureus types, respectively. A 61.71% of the MRSA isolates were genetically typed by PFGE resulting one type of MRSA with one genetic difference. Genome typing by PFGE was a powerful tool not only for strain identification but also for the resolution of clonal relationships of S. aureus strains. Observation on UV induction of S. aureus strains showed that the number of infective centers increased by more than 100%. As seen in our electron micrographs, our studied phages designated from ph1 to ph10; five of these phages ph2, ph4, ph5, ph7, and ph10 were morphotype A, present in Myoviredae group, their DNAs were in the range 43.6 kb-48.5 kb that is very close to this size range with minor differences in phage strain. Ph9 and ph10; had more than one band in their pattern. A mixture of ten phages differed in their virulence to MRSA isolates were used with all patient isolates (30) that were lysed completely by spot-test. Phage therapy for 30 patients of MRSA in different skin infection cases showed that phage therapy was highly effective. Using a mixture of 10 phages daily (7-10 days), treatment gave us a close success rate of 90%.