Subash Chandra Parija

Emergence of unusual species of enterococci causing infections, South India

BackgroundEnterococci tend to be one of the leading causes of nosocomial infections, with E. faecalis and E. faecium accounting up to 90% of the clinical isolates. Nevertheless, the incidence of other species of enterococci from clinical sources shows an alarming increase with the properties of intrinsic resistance to several antibiotics including beta-lactams and glycopeptides. Thus proper identification of enterococci to species level is quintessential for management and prevention of these bacteria in any healthcare facility. Hence this work was undertaken to study the prevalence of unusual species of enterococci causing human infections, in a tertiary care hospital in South India.MethodsThe study was conducted in a tertiary care hospital in South India from July 2001 to June 2003. Isolates of enterococci were collected from various clinical specimens and speciated using extensive phenotypic and physiological tests. Antimicrobial susceptibility testing were performed and interpreted as per NCCLS guidelines. Whole cell protein (WCP) fingerprinting of enterococci were done for species validation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed computationally.ResultsOur study showed the prevalence of unusual (non-faecalis and non-faecium enterococci) and atypical (biochemical variant) species of enterococci as 19% (46 isolates) and 5% (12 isolates) respectively. The 7 unusual species (46 isolates) isolated and confirmed by phenotypic characterization includes: 15 E. gallinarum (6.2%), 10 E. avium (4.1%), 6 E. raffinosus (2.5%), 6 E. hirae (2.5%), 4 E. mundtii (1.7%), 3 E. casseliflavus-including the two atypical isolates (1.2%) and 2 E. durans (0.8%). The 12 atypical enterococcal species (5%) that showed aberrant sugar reactions in conventional phenotyping were confirmed as E. faecalis, E. faecium and E. casseliflavus respectively by WCP fingerprinting. The antimicrobial susceptibility testing depicted the emergence of high-level aminoglycoside and beta-lactam resistance among different species apart from intrinsic vancomycin resistance by some species, while all the species tested were susceptible for linezolid and teicoplanin.ConclusionOur study reveals the emergence of multi-drug resistance among unusual species of enterococci posing a serious therapeutic challenge. Precise identification of enterococci to species level enables us to access the species-specific antimicrobial resistance characteristics, apart from knowing the epidemiological pattern and their clinical significance in human infections.

Molecular description of plasmid-mediated AmpC β-lactamases among nosocomial isolates of Escherichia coli & Klebsiella pneumoniae from six different hospitals in India

Background & objectives: Plasmid mediated AmpC β-lactamase (PMABL) resistance in Escherichia coli and Klebsiella spp. is an emerging problem worldwide. Phenotypic methods are commonly used for detection of PMABL production in Gram-negative isolates, but molecular data about the prevalence of plasmid-mediated AmpC-type resistance at the national level are needed. Hence, a prospective study was undertaken to determine the occurrence of PMABL gene and its types among clinical isolates of E. coli and K. pneumoniae obtained from six different hospitals in India. Methods: A total of 241 nosocomial isolates of K. pneumoniae (n=109) and E.coli (n=132) from six geographically distant hospitals in India were included. These were screened for cefoxitin resistance. AmpC disk test and modified three dimensional extraction test were used for phenotypic detection of PMABL production. Molecular types were determined by a multiplex PCR. Results: Among the 241 isolates, 187 (77.5%) were found to be cefoxitin resistant (K. pneumoniae n=83, E. coli n=104). AmpC activity was detectable in 153 (63.4%) isolates, (K. pneumoniae n=69, E. coli n=84). By PCR, the plasmid encoded AmpC genes were found in 92 (38.1%) isolates and the molecular types of the genes detected predominantly were DHA, CIT followed by MOX and ACC types. Interpretation & conclusions: A high percentage of plasmid-encoded AmpC enzymes was noted in E. coli and K. pneumonia isolates obtained from different parts of the country. Phenotypic methods alone may not reflect the true number of PMABL producers. Genotypic methods need to be employed in national surveillance studies.

A comparative study of blood smear, QBC and antigen detection for diagnosis of malaria.

Rapid diagnosis is prerequisite for effective treatment and reducing mortality and morbidity of malaria. This study was taken up to compare the efficacy of various methods available, i.e., thick and thin smear, quantitative buffy coat (QBC), plasmodium lactate dehydrogenase and aldolase in blood of patient. A total of 411 samples were collected from patients presenting with classic symptoms of malaria. For traditional microscopy; thick and thin smears were prepared and stained with Leishmans stain, taking thick smear as gold standard, thin smear had a sensitivity and specificity of 54.8% and 100%, respectively. QBC and antigen detection was done using commercially available kits; out of 411 samples, QBC and Malariagen were positive in 66 and 62 cases, with a sensitivity of 78% and 75%, respectively. Leishmans thick smear, although cost effective, is difficult to interpret for inexperienced microscopists; so if facilities are available, QBC should be used for routine diagnosis. In places where facilities are not available, rapid, simple and easy to interpret antigen detection test can be used despite low sensitivity.

Nosocomial cutaneous zygomycosis in a patient with diabetic ketoacidosis

Zygomycosis is an opportunistic fungal infection with a fulminant course. Varying clinical forms have been described, including cutaneous zygomycosis, which is mainly observed in diabetic and burns patients. We report herein a case of cutaneous zygomycosis of the nose in a 26-year-old female patient with diabetic ketoacidosis, developing secondary to the application of non-elasticized adhesive tape probably contaminated with fungal spores.

Efficacy of zinc supplementation on serum calprotectin, inflammatory cytokines and outcome in neonatal sepsis - a randomized controlled trial.

Abstract Objective: To find out the efficacy of zinc supplementation in decreasing the levels of serum calprotectin and inflammatory cytokines with improvement in outcome in neonatal sepsis. Methods: Neonates with clinical signs suggestive of sepsis and at least two screening tests positive were randomized into two groups – zinc group and control group. The zinc group received 3 mg/kg of zinc sulfate monohydrate twice a day orally for 10 days along with antibiotics. The control group received antibiotics and supportive care. Serum zinc, calprotectin, TNF-α and IL-6 were estimated in serum at recruitment and 10 days later after completion of antibiotics. The babies were monitored daily till discharge and mortality rate was compared between the groups. Results: Baseline characteristics were similar between the groups. Serum zinc levels were considerably increased in the zinc group after supplementation. There was significant decline in concentrations of serum calprotectin, TNF-α and IL-6 (p < 0.05) in the zinc group. In the control group also, serum calprotectin and IL-6 levels were found to be decreased significantly after antibiotic treatment (p < 0.05), while TNF-α showed insignificant reduction. Kaplan–Meier analysis was performed to assess the survival time between the groups. The mortality was lower in the zinc group compared to the control group 5 versus 11, p= 0.12. Conclusion: Neonates with sepsis who received zinc in addition to antibiotics showed significant reduction in serum calprotectin and inflammatory cytokines. Although mortality was lower in zinc group, it was not statistically significant.

Spectrum of vaginal discharge in a tertiary care setting

Introduction: Vaginal discharge is one of the common reasons for gynecological consultation. Many of the causes of vaginitis have a disturbed vaginal microbial ecosystem associated with them. Effective treatment of vaginal discharge requires that the etiologic diagnosis be established and identifying the same offers a precious input to syndromic management and provides an additional strategy for human immunodeficiency virus prevention. The present study was thus carried out to determine the various causes of vaginal discharge in a tertiary care setting. Materials and Methods: A total of 400 women presenting with vaginal discharge of age between 20 and 50 years, irrespective of marital status were included in this study and women who had used antibiotics or vaginal medication in the previous 14 days and pregnant women were excluded. Results: Of the 400 women with vaginal discharge studied, a diagnosis was established in 303 women. Infectious causes of vaginal discharge were observed in 207 (51.75%) women. Among them, bacterial vaginosis was the most common cause seen in 105 (26.25%) women. The other infections observed were candidiasis alone (61, 15.25%), trichomoniasis alone (12, 3%), mixed infections (22, 5.5%) and mucopurulent cervicitis (7 of the 130 cases looked for, 8.46%). Among the non-infectious causes, 72 (18%) women had physiological vaginal discharge and 13 (3.3%) women had cervical in situ cancers/carcinoma cervix. Conclusion: The pattern of infectious causes of vaginal discharge observed in our study was comparable with the other studies in India. Our study emphasizes the need for including Papanicolaou smear in the algorithm for evaluation of vaginal discharge, as it helps establish the etiology of vaginal discharge reliably and provides a valuable opportunity to screen for cervical malignancies.

Comparison of genomic DNA methylation pattern among septic and non-septic newborns - An epigenome wide association study.

DNA methylation is the current strategy in the field of biomarker discovery due to its prognostic efficiency. Its role in prognosis and early diagnosis has been recognized in various types of cancer. Sepsis still remains one of the major causes of neonatal mortality. Delay in diagnosis of sepsis leads to treatment difficulties and poor outcome. In this study, we have done an epigenome wide search to identify potential markers for prognosis of neonatal sepsis which may improve the treatment strategies. We analyzed the CpG methylation status in the epigenome of three septic and non-septic babies using Illumina Infinium HumanMethylation450K methylation microarray. The microarray data was analyzed with Illumina GenomeStudio v2011.1. After screening for biological and clinical significance, we found 81 differentially methylated CpGs located in 64 genes. Bioinformatic analysis using DAVID and GeneMania revealed a panel of differentially methylated protocadherin beta (PCDHB) genes that play vital role in leukocyte cell adhesion and Wnt signaling pathway. Apart, genes like CCS, DNAJA3, and DEGS2 were potentially hyper/hypo methylated which can be utilized in the development of novel biomarkers. This study will be helpful in exploring the role of DNA methylation in the pathophysiology of neonatal sepsis. The complete microarray data can be accessed from the public domain, Gene Expression Omnibus of NCBI (http://www.ncbi.nlm.nih.gov/geo/). The accession number is GSE58651.

Decreased baroreflex sensitivity is linked to sympathovagal imbalance, body fat mass and altered cardiometabolic profile in pre-obesity and obesity

OBJECTIVE Though decreased baroreflex sensitivity (BRS), the predictor of cardiac morbidities and mortality has been reported in obesity, the mechanisms and metabolic biomarkers influencing BRS have not been studied. We aimed to assess the difference in cardiovascular (CV) risk profile in pre-obesity and obesity, and the contribution of body composition and cardiometabolic factors to CV risks in these two conditions. METHODS Obesity indices, body composition, blood pressure variability and autonomic function test parameters were recorded in 223 subjects divided into controls (n=72), pre-obese (n=77) and obese (n=74) groups. Insulin resistance (HOMA-IR), atherogenic index (AI), leptin, adiponectin, inflammatory and oxidative stress parameters were measured. Association and independent contribution of altered cardiometabolic parameters with BRS were performed by Pearsons correlation and multiple regression analysis, respectively. RESULTS BRS was significantly decreased in pre-obese and obese group compared to controls. Sympathovagal imbalance (SVI) in the form of increased sympathetic and decreased parasympathetic cardiac drives was observed in pre-obesity and obesity. There was significant difference in general markers of obesity (body mass index, and waist-to-hip ratio), between pre-obese and obese group, however no such difference was observed in body composition and cardiometabolic parameters between the two groups. AI, high sensitive C-reactive protein (hs-CRP) and ratio of basal metabolism to body fat (BM/BF) in pre-obese group, and AI, HOMA-IR, leptin, adiponectin, ratio of basal metabolism to body weight (BM/BW), BM/BF, inflammatory and oxidative stress markers in obese group had independent contribution to BRS. Among these metabolic biomarkers, BRS had maximum association with leptin (β=0.532, p=0.000) in the obese group and hs-CRP (β=0.445, p=0.022) in the pre-obese group. CONCLUSIONS The present study demonstrates decreased BRS, an important marker of increased CV risk in pre-obesity and obesity. The intensity of cardiometabolic derangements and CV risk was comparable between pre-obese and obese subjects. BM/BF ratio appears to be a better marker of metabolic activity in pre-obesity and obesity. SVI and increased basal metabolism appear to be the physiological link between metabolic derangements and CV risks in both pre-obesity and obesity.

Diagnosis and management of human babesiosis.

Babesiosis is a protozoan parasitic infection affecting humans and animals. These infections are commonly transmitted by various species of Ixodes ticks depending upon the geographical location. They can also be transmitted by packed cell transfusion and transplacental route from mother to child. Various species have been reported to cause human infections, of which Babesia microti is the most common species reported globally. Usually, Babesia infections are asymptomatic or mild, but can be severe/life-threatening in immunosuppressed or splenectomized individuals. A high index of clinical suspicion in residents of endemic areas or individuals who had a recent travel history to such areas, with laboratory confirmation can guide an early institution of appropriate antimicrobial therapy, thereby preventing complications and death.

Serological and molecular diagnosis of hepatitis B virus

Background Hepatitis B is a potentially life threatening liver infection which leads to millions of deaths annually. Serological and molecular assays for hepatitis B virus are the major diagnostic tools. Dried blood spot (DBS), a minimally invasive procedure, is an alternative to serum and can be used for field based studies for molecular detection. The present aim of this study is to diagnose HBV infection by combination of serological and molecular methods from serum and dried blood spot.