ANTIMICROBIAL SUSCEPTIBILITY AND PREVALENCE OF EXTENDED SPECTRUM BETALACTAMASE (ESBL) AND METALLO BETALACTAMASE (MBL) AND ITS CO-EXISTENCE AMONG PSEUDOMONAS AERUGINOSA RECOVERED FROM OCULAR INFECTIONS

Objectives: To evaluate the changing trends in antimicrobial susceptibility rate and detection of ESBL and MBL among Pseudomonas aeruginosa isolated from various ocular infections over a 2 year periods with special reference to detection of ESBL and MBL co-existence among P aeruginosa recovered from ocular infections.Methods: All ocular specimens, culture positive for P aeruginosa (n=110) isolated from clinically suspected patients were submitted to L &T Microbiology Research Centre, Chennai, Tamil Nadu, India. Culture, antimicrobial susceptibility testing and ESBL detection was performed by Standard methods. MBL production was screened by Carbapenem-EDTA combination disk method.Results: Of the 3247 samples subjected to culture from August 2012– July 2014 by standard method 276 were positive for bacterial growth, thereby 8.5% of ocular infections mediated by bacterial pathogens. Out of 276 culture positives 110 (39.8%) Pseudomonas aeruginosa isolates recovered from ocular infections. The resistance rate for commonly used drugs against ocular infection includes Gentamycin [23.63%], Gatifloxacin [20.9%], Moxifloxacin [20%], Tobramycin [20%], ciprofloxacin [19.09%]. Totally 15 (13.63%) out of 110 isolates were identified as ESBL producer and 11 (10%) out of 110 isolates were identified as MBL producer by screening test, including 7 isolates have co-produced both ESBL and MBL enzymes and 4 isolates were only positive for MBL production.Conclusion: Though fluoroquinolones remains a good choice for ocular Pseudomonal infection. Gradual emergence of resistance to fluoroquinolones and aminoglycosides also noted from this study. The emergence of ESBL, MBL and pandrug resistance among P aeruginosa from ocular infections is an alarm rational finding which necessitates the earlier detection of both ESBL and MBL production as individual or co-existence in ocular isolates, which may pave the way for appropriate therapy for sight threatening conditions like endophthalmitis.Â

"pp65 antigenemia and real time polymerase chain reaction (PCR) based-study to determine the prevalence of human cytomegalovirus (HCMV) in kidney donors and recipients with follow-up studies."

<p>Abstract</p> <p>Background</p> <p>The present study was undertaken to determine the rate of occurrence of Human cytomegalovirus (HCMV) among kidney transplant recipients and donors by application of direct detection methods and to understand HCMV infection/disease development among transplanted patients as a prospective study.</p> <p>Results</p> <p>Peripheral blood samples collected from 76 kidney donors and 76 recipients from September 2007 to August 2009 were subjected to pp65 antigenemia and Quantitative real-time PCR (qRT-PCR) assays. Data were analyzed under Group A, B and C. Group A was further divided into sub-groups I, II, III, IV, and V for better understanding. Three, one and two donors in sub-group I, III, IV of Group A tested positive for real time PCR respectively. One recipient from group III tested positive for HCMV by qRT- PCR prior transplantation and remained positive one month post-transplantation. Three other recipients, tested negative prior to transplantation became positive a month after transplantation. Group B consisted of 18 donor-recipient pairs and one of the donor tested positive for HCMV by qRT-PCR. Eight recipients tested positive for HCMV one month after transplantation. The pp65 positivity and HCMV DNA load was high among group C recipients who mostly had symptoms of active disease. Significantly high values of pp65 antigenemia were observed among recipients of sub-group II (non-parametric chi-square test p = 0.007). Positive correlation between pp65 antigenemia and qRT-PCR value was observed. Thirty three of the recipients with disease treated with Valgancyclovir showed improved clinical outcome.</p> <p>Conclusion</p> <p>Our study showed that a significant proportion of kidney recipients develop HCMV infection following renal transplantation in spite of the absence of HCMV among donors. pp65 antigenemia assay and qRT- PCR methods can be applied to detect HCMV among kidney donors and recipients to monitor development of disease and these assays were predicative of HCMV infection among them. Clinical resistant to valganciclovir was not observed.</p

Nested reverse transcriptase–polymerase chain reactions targeting the messenger RNA of icl2, hspx, and rRNAP1 genes to detect viable Mycobacterium tuberculosis directly from clinical specimens

AbstractThere is an urgent need for a rapid and reliable test to detect actively multiplying Mycobacterium tuberculosis directly from clinical specimens for an early initiation of the appropriate antituberculous treatment. This study was aimed at the optimization and application of nested reverse transcriptase–PCR (nRT–PCR) targeting the messenger RNA of the icl2, hspx, and rRNAP1 genes directly from sputum specimens, and their evaluation against the culture by the BACTEC MicroMGIT mycobacterial culture system. 203 Sputum samples from clinically suspected tuberculosis patients and 30 control specimens (clinically proven viral or bacterial infections other than tuberculosis) were included in this study. The mycobacterial culture was performed by the BACTEC MicroMGIT system following the manufacturer’s instructions. The primers for nRT–PCRs targeting icl2, hspx, and rRNAP1 genes were indigenously designed using the Primer-BLAST software, and optimized for sensitivity and specificity. The icl2, hspx, and rRNAP1 genes were able to pick up 63.9%, 67.2%, and 58.75%, respectively, of culture-negative sputum specimens collected from clinically suspected tuberculosis patients. However, three (1.4%) were negative for nRT–PCR, but M. tuberculosis culture positive. All the 30 controls were negative for culture by the BACTEC MicroMGIT method and all three nRT–PCR. The novel nRT–PCRs targeting icl2, hspx, and rRNAP1 genes developed in this study are rapid and reliable diagnostic tools to detect viable M. tuberculosis directly from sputum specimens. However, further study by including a larger number of sputum specimens needs to be carried out to ascertain the diagnostic utility of the novel nRT–PCRs optimized in the study

Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

ABSTRACT Aim: Isolation, dark field detection and microscopic agglutination test (MAT) are considered -gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR) -based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septicemia. Methodology and Results: Two hundred and seven (207) blood samples from patients were diagnosed with septicemia which includes 100 bacterial (other than Leptospira) culture positive and 107 bacterial culture negative samples were studied. Primers for Nested PCR targeting LipL32 gene of Leptospira interrogans were designed and the specificity of primers was tested against serum samples positive/negative by either MAT or dark field microscopy. PCR amplified products were further confirmed by DNA sequencing. The standardized nPCR was sensitive and specific to Leptospira interrogans. Twenty-one (21%) out of 100 culture positive blood samples, three (2.8%) out of 107 culture negative samples showed nPCR positivity and were confirmed as Leptospira interrogans by DNA sequencing (p&lt;0.001). A sensitive nPCR specific to Leptospira interrogans was developed. Conclusion, significance and impact of study: The p value (&lt;0.001) signifies that Leptospira is commonly associated with other bacteria circulating in blood indicating that a decreased immune status is created primarily by a bacterium with enhanced possibility of development of Leptospiral infection probably be of an endogenous origin

Genomic analysis of a large set of currently—and historically—important human adenovirus pathogens

Human adenoviruses (HAdVs) are uniquely important “model organisms” as they have been used to elucidate fundamental biological processes, are recognized as complex pathogens, and are used as remedies for human health. As pathogens, HAdVs may effect asymptomatic or mild and severe symptomatic disease upon their infection of respiratory, ocular, gastrointestinal, and genitourinary systems. High-resolution genomic data have enhanced the understanding of HAdV epidemiology, with recombination recognized as an important and major pathway in the molecular evolution and genesis of emergent HAdV pathogens. To support this view and to actualize an algorithm for identifying, characterizing, and typing novel HAdVs, we determined the DNA sequence of 95 isolates from archives containing historically important pathogens and collections housing currently circulating strains to be sequenced. Of the 85 samples that were completely sequenced, 18 novel recombinants within species HAdV-B and D were identified. Two HAdV-D genomes were found to contain novel penton base and fiber genes with significant divergence from known molecular types. In this data set, we found additional isolates of HAdV-D53 and HAdV-D58, two novel genotypes recognized recently using genomics. This supports the thesis that novel HAdV genotypes are not limited to “one-time” appearances of the prototype but are of importance in HAdV epidemiology. These data underscore the significance of lateral genomic transfer in HAdV evolution and reinforce the potential public health impact of novel genotypes of HAdVs emerging in the population

The diagnostic significance of enzyme linked immuno-sorbent assay for herpes simplex, varicella zoster and cytomegalovirus retinitis.

<b>Purpose:</b> To evaluate the diagnostic usefulness of enzyme linked immuno-sorbent assay (ELISA) in single serum samples to associate herpes simplex virus (HSV), varicella zoster virus (VZV) or cytomegalovirus (CMV) with viral retinitis as against polymerase chain reaction (PCR) on intraocular specimens. It was also designed to study the seroprevalence in normal healthy individuals, and the genomic prevalence of HSV, VZV and CMV in patients without an active viral inflammatory process. <b> Methods: </b> PCR for the detection of HSV, VZV and CMV genomes was done on 33 and 90 intraocular fluids from viral retinal patients and non-viral controls respectively. ELISA was done on 30 and 100 serum samples from viral retinitis patients and normal healthy controls respectively. <b> Results:</b> PCR did not detect HSV, VZV and CMV genomes except one, in which VZV-DNA was detected. ELISA showed prevalence rates of 28&#x0025;, 83&#x0025; and 90&#x0025; for antibodies against HSV, VZV and CMV respectively in the normal population. In the 30 viral retinitis patients, PCR detected HSV-DNA in 2 (6.7&#x0025;), VZV-DNA in 7 (23.3&#x0025;) and CMV-DNA in 6 (20.0&#x0025;) patients, while ELISA detected antibodies against HSV, VZV and CMV in 13 (43.3&#x0025;), 24 (80.0&#x0025;) and 23 (76.7&#x0025;) patients respectively. ELISA was of value in indirect diagnosis only in 6 (20.0&#x0025;) as compared to 15 (50.0&#x0025;) of 30 patients by PCR, this difference was statistically significant (McNemar test, P value = 0.005). <b> Conclusion: </b> Serology by ELISA is no longer a useful diagnostic tool to associate HSV, VZV and CMV viruses with viral retinitis

Effects of viscoelastic ophthalmic solutions on cell cultures

The development of mild but significant inflammation probably attributable to viscoelastic ophthalmic solutions in cataract surgery was recently brought to the notice of the authors, and hence a study of the effects of these solutions available in India, on cell cultures was undertaken. We studied the effects of 6 viscoelastic ophthalmic solutions (2 sodium hyaluronate designated as A and B, and 4 hydroxypropylmethylcellulose designated as C, D, E and F) on HeLa, Vero and BHK-21 cell lines in tissue culture microtitre plates using undiluted, 1:10 and 1:100 dilutions of the solutions, and in cover slip cultures using undiluted solutions. Phase contrast microscopic examination of the solutions was also done to determine the presence of floating particles. The products D and F produced cytotoxic changes in HeLa cell line and these products also showed the presence of floating particles under phase contrast microscopy. Other products did not have any adverse effects on the cell lines nor did they show floating particles. The viscoelastic ophthalmic pharmaceutical products designated D and F have cytotoxic effects on HeLa cell line which appears to be a useful cell line for testing these products for their toxicity. The presence of particulate materials in products D and F indicates that the methods used for purification of the solution are not effective