Phenotypic & genotypic profile of antimicrobial resistance in Pseudomonas species in hospitalized patients.

Background & objectives: Nosocomial infections caused by multidrug-resistant, Pseudomonas species have become a major clinical and public health concern. The aim of this study was to characterize phenotypic and genotypic profile of antimicrobial resistance (AMR) in Pseudomonas spp. isolated from hospitalized patients. Methods: A total of 126 consecutive, non-duplicate isolates of Pseudomonas spp. isolated from various clinical samples were included in the study over a period of two years. Identification and antimicrobial sensitivity was performed using automated culture system according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. Phenotypic detection of extended-spectrum ß-lactamases (ESBLs), Amp-C ß-lactamase (AmpC) and metallo-ß-lactamases (MBLs) were done by various combinations of disc-diffusion and E-test methods, followed by polymerase chain reaction-based detection of ß-lactamase-encoding genes. Results: Among 126 clinical isolates, 121 (96.1%) isolates were identified as Pseudomonas aeruginosa. Most of the isolates were recovered from pus sample, 35 (27.8%) followed by urine, 25 (19.84%); endotracheal aspirate, 24 (19.04%); blood, 14 (11.11%) and sputum, four (3.17%). The highest rate of resistance was against ticarcillin-clavulanic acid, 113 (89.7%) followed by meropenem, 92 (72.5%) and ceftazidime, 91 (72.3%). Overall, ESBLs, AmpC and carbapenemase production was detected in 109 (96.4%), 64 (50.8%) and 105 (94.6%) isolates by phenotypic methods. The most prevalent ESBL gene was blaTEMin 72 (57.1%) and the least prevalent was blaSHVin 19 (15.1%) isolates. AmpC gene was seen less compared to ESBL gene. The most prevalent carbapenemases gene was blaNDM-141 (46.06%) followed by blaVIM and blaOXA-1. Interpretation & conclusions: Our findings suggested that a high rate of ESBLs and carbapenemases production was observed in Pseudomonas spp. Therefore, phenotypic and genotypic detection of AMR needs to be combined for better characterization of resistance patterns in Pseudomonas spp.

Epidemiological trends of fatal pediatric trauma: A single-center study.

To evaluate the potential risk factors which increase the incidence of post-trauma complications and mortality in pediatric population.A retrospective cohort study was conducted on patients below 18 years of age with a fatal outcome who were admitted to an Indian level-1 trauma center between January 2013 and December 2015. This cohort was analyzed to determine the demographics, injury mechanism, injury severity, microbiological profile, and cause of death.In total, 320 pediatric patients with a fatal outcome were studied which showed male preponderance (71.56%). The median age of the patients was 11 years (range, 0.14-18 years). Median duration of stay was 1 day (range, 0-183 days). Fall and road traffic accidents were the common mechanisms of trauma while the main injury was head injury. In total, 857 clinical samples were received from 56 patients. The clinical samples from 35 (10.94%) patients were culture positive. Culture-proven infections were significantly correlated with the length of hospital stay (P = .001). In total, 212 organisms were isolated from 193 positive samples of which gram-negative bacteria were predominant (89.15%). The most common gram-positive bacterial isolate was Staphylococcus aureus (12, 52.17%), while Acinetobacter baumannii (66, 34.92%) was the most prevalent gram-negative bacterial isolate followed by Pseudomonas spp. (36, 19.05%), Klebsiella pneumoniae (35, 18.52%), and Escherichia coli (16, 8.47%). Up to 100% multidrug resistance was seen in both gram-positive and gram-negative bacterial isolates. The first 24 hours after trauma were the deadliest for our patients. Head/central nervous system injury was the primary cause of disabilities and early death whereas infection attributed to prolonged hospital stay.From these observations we concluded that management of pediatric trauma requires expert, multidisciplinary, and timely interventions. Moreover, nosocomial infections with multidrug resistant gram-negative bacteria challenges the accepted tenets of trauma care affecting the outcome of the pediatric population. Early identification of such high-risk patients' infection may facilitate early intervention. Thus, many deaths in pediatric group are preventable.

Magnetic resonance imaging for tumor restaging after chemotherapy in retinoblastoma with optic nerve invasion.

PURPOSE: Extraocular retinoblastoma with optic nerve invasion is treated by a multimodal protocol consisting of neoadjuvant chemotherapy, enucleation, and adjuvant therapy. This study was conducted to evaluate the performance of magnetic resonance imaging (MRI) used for tumor restaging in these children after systemic chemotherapy administration. METHODS: Contrast-enhanced MRI scan of orbits and brain was performed at diagnosis and patients were treated with neoadjuvant chemotherapy. After chemotherapy, MRI scan was repeated for tumor restaging and residual post-laminar thickening and/or enhancement of the affected optic nerve, if any, was recorded. MRI findings were correlated with histopathology in enucleated specimens. The main outcome measures were specificity, sensitivity, and accuracy of MRI in predicting post-laminar invasion after neoadjuvant chemotherapy. RESULTS: A total of 46 eyes (46 patients) were studied. Optic nerve thickening on MRI had a sensitivity, specificity, and accuracy of 100% (95% Confidence Interval (CI): 64.6-100%), 76.9% (95% CI: 61.7-87.4%), and 80.4% (95% CI: 66.8-89.4%), respectively. Optic nerve enhancement had a sensitivity, specificity, and accuracy of 85.7% (95% CI: 48.7-97.4%), 79.5 % (95% CI: 64.5-89.2%), and 80.4% (95% CI: 66.8-89.4%), respectively. Combined thickening and enhancement of the optic nerve had a sensitivity, specificity, and accuracy of 100% (95% CI: 60.9-100%), 82.4% (95% CI: 66.5-91.7%), and 85% (95% CI: 70.9-92.9%), respectively. CONCLUSION: MRI is a valuable tool for restaging of retinoblastoma and predicting residual optic nerve disease after neoadjuvant chemotherapy. Combined thickening and enhancement on MRI appeared to be a more reliable indicator of post-laminar invasion as compared to thickening or enhancement alone.

Five-year profile of candidaemia at an Indian trauma centre: High rates of Candida auris blood stream infections.

Candidaemia is a potentially fatal infection with varied distribution of Candida species and their antifungal susceptibility profiles. The recent emergence of Candida auris in invasive candidiasis is a cause for concern. This study describes the profile of candidaemia at an Indian tertiary care hospital and reports the emergence of C. auris. All patients diagnosed with candidaemia between 2012 and 2017 were studied. The isolates were identified using conventional methods, VITEK 2 and MALDI-TOF MS. The isolates not identified by MALDI-TOF were sequenced. Antifungal susceptibility testing was done by the CLSI broth microdilution method and VITEK 2. A total of 114 isolates of Candida species were analysed. Candida tropicalis (39.4%) was the most common species, followed by C. auris (17.5%), C. albicans (14%) and C. parapsilosis (11.4%). Notably, Diutina mesorugosa isolates (n = 10) were not identified by MALDI-TOF and were confirmed by sequencing. Furthermore, 45% (n = 9) C. auris strains exhibited low MICs of FLU (0.05-4 µg/mL) and the remaining 55% (n = 11) isolates had high MICs ≥ 64 µg/mL. Also, D. mesorugosa exhibited high MICs of FLU (32 µg/mL) in 2 isolates. A high rate of errors in antifungal susceptibility was noted with the VITEK 2 as compared to the CLSI method. Candida auris was the second most prevalent species causing candidaemia warranting infection control practices to be strengthened to prevent its spread.

Multimodal Therapy for Stage III Retinoblastoma (International Retinoblastoma Staging System): A Prospective Comparative Study.

PURPOSE: To compare the efficacy of 2 chemotherapeutic drug combinations as part of multimodal therapy for orbital retinoblastoma. DESIGN: Prospective, comparative, study. PARTICIPANTS: Patients with stage III retinoblastoma (International Retinoblastoma Staging System). METHODS: Demographic and clinical features were recorded at presentation. Treatment consisted of a multimodal protocol with neoadjuvant chemotherapy, enucleation, orbital external-beam radiotherapy, and adjuvant chemotherapy. For chemotherapy, patients were randomized into 2 groups: group A patients were treated with vincristine, etoposide, and carboplatin (VEC) and group B patients were treated with carboplatin and etoposide, alternating with cyclophosphamide, idarubicin, and vincristine. Treatment outcomes and adverse effects were recorded. Efficacy parameters were compared between the groups. MAIN OUTCOME MEASURES: Survival probability, cause of death, and chemotherapy-related toxicity. RESULTS: A total of 54 children were recruited (27 in each group). The mean ± SD follow-up was 21.3±11.34 months. The overall Kaplan-Meier survival probability was 80% (95% confidence interval [CI], 0.67-0.89) and 42% (95% CI, 0.24-0.59) at 1 year and 4 years, respectively. There were 9 deaths in group A and 15 deaths in group B. The Kaplan-Meier survival probability at 1 year was similar between the groups: 81% (95% CI, 0.60-0.91) and 79% (95% CI, 0.58-0.9) for groups A and B, respectively. At 4 years, the survival probability for group A was higher (63% [95% CI, 0.41-0.79] vs. 25% [95% CI, 0.08-0.46] for groups A and B, respectively), with a strong trend of better survival in group A over time (P = 0.05). The major cause of death was central nervous system relapse (8 patients in group A and 7 patients in group B). Two patients in group B died of sepsis after febrile neutropenia. Grade 3 and grade 4 hematologic toxicities were more common in group B, with a significant difference in grade 4 neutropenia (P = 0.002). CONCLUSIONS: This study compared the outcomes of VEC chemotherapy with a 5-drug combination of etoposide and carboplatin, alternating with cyclophosphamide, idarubicin, and vincristine, for stage III retinoblastoma. The VEC combination was found to be more effective and may be recommended as neoadjuvant and adjuvant chemotherapy.

Clinical presentation and survival of retinoblastoma in Indian children.

OBJECTIVE: To study the clinical presentation and survival among Indian children with retinoblastoma (RB) and to determine factors predictive of poor outcome. METHODS: A retrospective review of children newly diagnosed with RB at a tertiary referral centre was undertaken. Demographic and clinical characteristics and treatment outcomes were studied. RESULTS: A total of 600 patients (unilateral 67.6%, bilateral 32.4%) was studied. 61% was boys. The median age at presentation was 29 months (18 months vs 36 months in bilateral and unilateral cases, respectively, p<0.001). leukocoria was most common (83%), followed by proptosis (17%). Tumours were intraocular in 72.3% and extraocular in 27.7% cases. In the intraocular group, 78% were advanced Group D or E disease. Metastasis to the central nervous system was noted in 15.7% of extraocular cases. A statistically significant difference was seen between intraocular and extraocular groups in the median age (24 months vs 37.5 months, p<0.001) and median lag period (2.5 months vs 7 months, p<0.001). The Kaplan-Meier survival probability was 83%, 73% and 65% at 1 year, 2 years and 5 years, respectively. On univariate analysis, age >2 years (p=0.002), lag period >6 months (p=0.004) and extraocular stage (p<0.001) were associated with poor outcome. On multivariate analysis, extraocular invasion was predictive of low survival (HR 5.04, p<0.001). CONCLUSIONS: Delayed presentation is a matter of concern. Improving awareness about the early signs and creating facilities for diagnosing and treating RB at the primary and secondary levels of healthcare are required to reduce mortality and morbidity, and lead to improved outcomes that are comparable with the developed nations.

Isolation and characterization of senescent Cryptococcus neoformans and implications for phenotypic switching and pathogenesis in chronic cryptococcosis.

Although several virulence factors and associated genes have been identified, the mechanisms that allow Cryptococcus neoformans to adapt during chronic infection and to persist in immunocompromised hosts remain poorly understood. Characterization of senescent cells of C. neoformans demonstrated that these cells exhibit a significantly enlarged cell body and capsule but still cross the blood-brain barrier. C. neoformans cells with advanced generational age are also more resistant to phagocytosis and killing by antifungals, which could promote their selection during chronic disease in humans. Senescent cells of RC-2, a C. neoformans strain that undergoes phenotypic switching, manifest switching rates up to 11-fold higher than those of younger cells. Infection experiments with labeled cells suggest that senescent yeast cells can potentially accumulate in vivo. Mathematical modeling incorporating different switching rates demonstrates how increased switching rates promote the emergence of hypervirulent mucoid variants during chronic infection. Our findings introduce the intriguing concept that senescence in eukaryotic pathogens could be a mechanism of microevolution that may promote pathoadaptation and facilitate evasion of an evolving immune response.

Biofilm formation in clinical Candida isolates and its association with virulence.

Biofilm formation, an important virulence trait of Candida species was measured in 107 Candida isolates from 32 candidemic patients by XTT [2,3-bis (2-methoxy-4nitro-5-sulfo-phenyl)-2H-tetra-zolium-5-carboxanilide] activity and compared to biofilm formation of Candida isolates from oropharyngeal lesions of 19 AIDS patients. Biofilm formation by XTT varied among species and C. albicans; C. lusitaniae and C. krusei produced more biofilm than the other Candida species. C. tropicalis was the most dominant species isolated from blood followed by C. albicans, and other non-albicans species whereas only C. albicans was recovered from oral lesions. Importantly, though Biofilm formation was variable within a species it was stable in sequential isolates during chronic infection. Sequential isolates exhibited identical Karyotype pattern or RAPD patterns unless patients were co-infected with more than one strain. High biofilm formation was associated with slow growth rate but not with adherence. Murine infection studies demonstrated that, degree of in-vitro biofilm formation was associated with virulence in mice, as mice infected both with no and low biofilm formers survived longer than mice infected with high biofilm former C. albicans (p< or =0.001). We conclude that biofilm formation is a stable but strain specific characteristic that can greatly vary among C. albicans and non-albicans strains, and plays an important role in persistence of infection.

Phenotypic Switching in Fungi.

Over the past three decades new fungal diseases have emerged that now constitute a major threat, especially for patients with chronic diseases and/or underlying immune defi ciencies. Despite the epidemiologic data, the emergence of stable drug-resistant or hyper-virulent fungal strains in human disease has not been demonstrated as seen in emerging viral and bacterial infections. Fungi are eukaryotic microbes that capitalize on a sophisticated built-in ability to generate phenotypic variability. This successful strategy allows them to undergo rapid adaptation in response to environmental challenges, such as individual body locations that may exhibit drastic differences in temperature and pH. Rapid microevolution can also confer drug resistance and protect them from the host's immune response. This review explores phenotypic switching in pathogenic fungi, including Candida spp and Cryptococcus spp, and how phenotypic switching contributes to the pathogenesis of fungal diseases.

Prevalence & susceptibility to fluconazole of Candida species causing vulvovaginitis.

BACKGROUND & OBJECTIVE: Vulvovaginal candidiasis is an important cause of morbidity in women of reproductive age. This study was carried out to determine the species prevalence and susceptibility pattern to fluconazole of yeasts isolated from the vagina of symptomatic women. METHODS: This prospective study was conducted in a rural primary health care center of north India from May 2003 to April 2004 and included 601 married, sexually active women (18-49 yr) with the self reported symptoms of vaginal discharge and/or genital itching and/or genital burning. Specific aetiology of the genitourinary symptoms including candidal infection were determined. Specimens from the lateral wall of vagina were subjected to direct wet mount microscopy and fungal culture on Sabouraud's dextrose agar. Susceptibility testing to fluconazole was carried out using broth microdilution method. RESULTS: Yeasts were isolated in 111 (18.5%) women and these consisted of Candida glabrata (56, 50.4%), C. albicans (39, 35.1%), C. tropicalis (12, 10.8%), C. krusei (3, 2.7%) and C. parapsilosis (1, 0.9%). Susceptibility testing carried out on 30 representative isolates (15 C. glabrata, 10 C. albicans, 4 C. tropicalis and 1 C. parapsilosis) revealed that 21 isolates (70%) were susceptible (MIC, < or = 8 microg/ml) to fluconazole while 9 (30%) were susceptible-dose dependent (S-DD, MIC 16-32 microg/ml). INTERPRETATION & CONCLUSION: Our findings suggest a low prevalence of fluconazole resistance in vaginal candida isolates in our population. However, a high prevalence of non-albicans candida species and increased dose-dependent resistance in these isolates necessitates vigilance since this may warrant a change in the optimal therapy of non-albicans candida vaginitis.