Bijayini Behera

An evaluation of four different phenotypic techniques for detection of metallo-β-lactamase producing Pseudomonas aeruginosa

PURPOSE The present study was undertaken to detect metallo-beta-lactamase (MBL) in nosocomial isolates of Pseudomonas aeruginosa by four different phenotypic methods. METHODS Ninety-one consecutive P. aeruginosa isolates were subjected to susceptibility testing by disc-diffusion assay and Vitek 2. Imipenem resistance was determined by three different methods (disc-diffusion, Vitek 2 and E test). Screening for MBL production was done by imipenem-EDTA combined disc test, imipenem-EDTA double-disc synergy test, imipenem-EDTA MBL E test and EDTA disc potentiation using four cephalosporins. RESULTS Of 63 imipenem resistant isolates, MBL screening could be done in 56 isolates, of which 48 were MBL positive by combined disc test and 36 by the double disc synergy test. For confirmation of MBL production, MBL E test was done in 30 isolates. All the 30 isolates were confirmed to be MBL positive by the MBL E test method. EDTA disc potentiation using four cephalosporins was not very useful for MBL detection. CONCLUSIONS Imipenem-EDTA combined disc test and imipenem-EDTA MBL E test are equally effective for MBL detection, but given the cost-constraints, combined disc test can be used as a convenient screening method in the clinical microbiology laboratory.

Evaluation of susceptibility testing methods for polymyxin

BACKGROUND The widespread resistance in Gram-negative bacteria has necessitated evaluation of the use of older antimicrobials such as polymyxins. In the present study we evaluated the different susceptibility testing methods for polymyxins B and E against Gram-negative bacteria using the new Clinical and Laboratory Standards Institute (CLSI) guidelines. METHODS The susceptibility of 281 multidrug-resistant (MDR) Gram-negative bacteria (GNB) to polymyxin B was evaluated, comparing broth microdilution (BMD; reference method), agar dilution, E-test, and disk diffusion. Disk diffusion testing of polymyxin B was also performed against 723 MDR GNB. RESULTS Twenty-four of 281 (8.5%) isolates were found to be resistant to polymyxin B by the reference BMD method. The rates of very major errors for agar dilution and E-test (for polymyxin B) were 0.7% and 1%, respectively, and those for disk diffusion (for polymyxin B and polymyxin E) were 1% and 0.7%, respectively. For the 257 isolates found sensitive by reference BMD, the rates of major errors by agar dilution and E-test (for polymyxin B) were 2.4% and 0%, respectively, and those for disk diffusion (polymyxin B and polymyxin E) were 0% and 0.7%, respectively. Twenty-six (3.6%) of the 723 Gram-negative isolates were resistant to polymyxin B by disk diffusion. CONCLUSION The E-test and agar dilution methods showed good concordance with BMD. The disk diffusion method can be useful for initial screening in diagnostic laboratories.

Post-traumatic skin and soft tissue infection due to Aeromonas hydrophila.

We report a case of posttraumatic skin and soft tissue infection in a patient who sustained laceration after being hit by a water tanker. Aeromonas hydrophila was isolated from pus and was identified to the species level by Vitek 2 and a battery of biochemical tests. The patient responded to thorough drainage, debridement of wound and 2 weeks of intravenous antibiotics. The patient was taken up for split skin grafting of the raw area. She was discharged with satisfactory graft uptake after 1 week without any further antibiotics advice. Follow-up after 3 weeks was satisfactory with healthy cover on the raw area and normal weight bearing on the left leg.

Rapid identification of yeast isolates from clinical specimens in critically Ill trauma ICU patients

Purpose: The purpose was to evaluate the performance of a commercially available chromogenic Candida speciation media and the Vitek 2 ID system for the identification of medically important yeasts and yeast-like organisms in a routine clinical microbiology laboratory. Materials and Methods: A total of 429 non duplicate, consecutive yeast strains were included during the 3.5-year study period. The performance of the Vitek 2 ID system and a chromogenic agar medium was evaluated against the gold standard conventional phenotypic and biochemical identification method for speciation of yeast isolates from trauma patients. Results: Candida tropicalis (64%) was the most common Candida species, followed by Candida albicans (14%), Candida rugosa (7%), and Candida parapsilosis (6.5%). Of the 429 isolates, 183 could be identified to species level by all the three methods. Agreement between the chromogenic agar method and conventional methods was 80% for Candida tropicalis, 100% for Candida rugosa, 89% for Candida albicans, and 77% for Candida parapsilosis. Vitek 2 had lower sensitivity, with agreement of 49% for Candida tropicalis, 100% for Candida rugosa, 39% for Candida albicans, and 31% for Candida parapsilosis. Conclusion: Thus, in long-term ICU patients, an increasing trend of isolating nonalbicans Candida spp. continues. The chromogenic agar medium is a convenient and economic method to identify commonly isolated species in busy clinical microbiology laboratories.

Candida rugosa: a possible emerging cause of candidaemia in trauma patients

IntroductionCandida rugosa appears to be emerging as a distinctive cause of candidaemia in recent years. Candidaemia due to this species is important to recognise because of its decreased susceptibility to azoles.Materials and methodsWe retrospectively evaluated a cluster of C. rugosa candidaemia occurring in critically ill trauma patients from a level I trauma centre of India. During the period from July 2008 to September 2009, a total of 28 blood samples from 19 patients were found to be positive for C. rugosa. Genetic relatedness among 17 C. rugosa isolates were characterised by the random amplified polymorphic DNA (RAPD) assay using M13 primers. These isolates were also characterised for their susceptibility to four antifungal agents, amphotericin B, fluconazole, flucytosine and voriconazole.ResultsIn our study, 21% of C. rugosa isolates were resistant to fluconazole, whereas 100% susceptibility to amphotericin B, flucytosine and voriconazole was noted. Thirteen out of the 19 patients (68.4%) with C. rugosa candidaemia died. Of these, six had received antifungal therapy after confirmation of fungaemia.DiscussionPrior to this cluster, C. rugosa had never been identified as a cause of infection at our centre. Due to the retrospective nature of the evaluation of these cases, the source of this possible outbreak could not be traced. Nevertheless, to the best of our knowledge, this is the largest cluster of cases of C. rugosa candidaemia reported from a single institution in the English literature.

Salmonella enterica Enteritidis Arthritis Following Trauma in a Child with Thalassemia Major

Osteoarticular infections caused by Non-typhi Salmonella are exceptionally encountered. We report a case of a bacteriologically documented knee joint infection due to Salmonella enterica serotype enteritidis, following trauma in a child with thalassemia major. Emergency arthrotomy combined with antimicrobial therapy was helpful in eradication of infection. Physicians should be aware of this rare manifestation of Non-typhi Salmonella infections in thalassemic patients.

Resistance pattern of mupirocin in methicillin-resistant Staphylococcus aureus in trauma patients and comparison between disc diffusion and E-test for better detection of resistance in low resource countries

Introduction: Mupirocin is an effective antibiotic for elimination of methicillin-resistant Staphylococcus aureus (MRSA) from nasal colonization and has been used to control outbreaks. Current reports show an increasing trend of resistance to this antibiotic. Objective: This study was conducted to analyze the resistance pattern of MRSA to mupirocin among the patients admitted following trauma to an apex trauma care center of India and to compare the efficacy between two methods of antimicrobial sensitivity testing. Materials and Methods: A total of 150 isolates of MRSA from various clinical samples of trauma patients over a period of 2 years were included in this study. These strains were confirmed for MRSA using VITEK® 2 Compact and the Clinical Laboratory Standard Institute disc diffusion methods. The mupirocin susceptibility of the strains was tested by using E-test and 5 μg mupirocin disc in parallel each time, and the results were compared. Results: Clear zones of inhibition were observed in both tests. Though, good correlation was observed between the disc diffusion and E-tests in >98%, E-test showed a tendency to show lower minimum inhibitory concentration (MIC) in the remaining. These finding did not affect the final interpretation or outcomes. Of the total 150 strains, 138 (92%) showed sensitivity with the zone size in the range of 30-45 mm by 5 μg disc; rest (8%) showed sensitivity with the zone in the range of 18-30 mm by 5 μg disc, but 143 (95%) showed MIC ≤ 0.094 μg/ml and 8 (5%) gave MIC ≤ 0.75 μg/ml but ≥0.094 μg/ml by E-test. However, when both tests were compared, 5 (3.3%) showed zone size between 14 and 25 mm with ≤0.75 but >0.25 μg/ml MIC; 7 (5%) falling between 25 and 30 mm zone with MIC of ≤0.25 but >0.094 μg/ml and 138 (92%) showed zone >30 mm with MIC ≤0.094 but >0.064 μg/ml. Conclusions: All the MRSA isolates in our study were sensitive to mupirocin which is an encouraging finding. Though good screening for sensitivity can be done with 5 μg mupirocin disc, E-test provides a much clear and accurate results in clinical set-up. Hence, disc test can be used in resource poor countries and supplemented with E-test when needed.

Clinical and molecular epidemiology of beta-hemolytic streptococcal infections in India.

INTRODUCTION Beta-hemolytic streptococci (βHS) cause a diverse array of human infections. Despite the high number of cases of streptococcal carriers and diseases, studies discerning the molecular epidemiology of βHS in India are limited. This study reports the molecular and clinical epidemiology of beta-hemolytic streptococcal infections from two geographically distinct regions of India. METHODOLOGY A total of 186 isolates of βHS from north and south India were included. The isolates were identified to species level and subjected to antimicrobial susceptibility testing. Polymerase chain reaction (PCR) was done to detect exotoxin genes, and emm types of group A streptococci (GAS) strains were ascertained by sequencing. RESULTS GAS was the most common isolate (71.5%), followed by group G streptococci (GGS) (21%). A large proportion of GAS produced speB (97%), smeZ (89%), speF (91%), and speG (84%). SmeZ was produced by 21% and 50% of GGS and GGS, respectively. A total of 45 different emm types/subtypes were seen in GAS, with emm 11 being the most common. Resistance to tetracycline (73%) and erythromycin (34.5%) was commonly seen in GAS. CONCLUSIONS A high diversity of emm types was seen in Indian GAS isolates with high macrolide and tetracycline resistance. SpeA was less commonly seen in Indian GAS isolates. There was no association between disease severity and exotoxin gene production.

Evaluation of Loop mediated isothermal amplification (LAMP) assay in the diagnosis of Tubercular lymphadenitis: A pilot study

Tubercular lymphadenitis (TBLA) contributes to 30-40% of extrapulmonary TB cases in the immunocompetent individuals and 40-50% in people with HIV. Current diagnostic methods for TBLA like Gene-Xpert or PCR are costly and conventional methods like fine needle aspiration cytology, histopathology lack sensitivity and specificity. Culture which is considered as gold standard require high turnaround time. Loop mediated isothermal amplification (LAMP) assay has been developed as a novel technique for nucleic acid amplification and has shown promising results in the diagnosis of pulmonary tuberculosis. Present study evaluated the Nu-LAMPTM TB Kit (RAS Life Sciences Pvt. Ltd, a bioMerieux group company) for diagnosis of TBLA comparing with conventional tests (cytology, ZN smear, culture). The sensitivity, specificity, PPV and NPV of LAMP assay was found to be 33.3%, 91.2%, 40% and 88.57% as compared to 100%, 76.5%, 42.9% and 100% of ZN staining and 100%, 73.5%, 40% and 100% of cytopathology. The low sensitivity of LAMP assay in the present study addresses the need for comparison and validation of the commercially available LAMP kits before used for patient diagnosis.

Antimicrobial resistance in beta-haemolytic streptococci in India: A four-year study

Background & objectives: The incidence and severity of invasive and non-invasive infections demonstrate variability over time. The emerging resistance of Group A streptococci (GAS) to commonly used antibiotics is of grave concern. This study was conducted to assess the antimicrobial resistance of beta-haemolytic streptococci (βHS) in India and to ascertain the molecular mechanisms of resistance. Methods: All isolates of βHS from the Trauma Centre of All India Institute of Medical Sciences (AIIMS) (north India), and heavily populated area of old Delhi from 2010 to 2014 and Yashoda Hospital, Secunderabad (in south India, 2010-2012) and preserved isolates of βHS at AIIMS (2005-2009) were included. Phenotypic confirmation was done using conventional methods and the Vitek 2. Antibiotic sensitivity testing was done by disc diffusion and E-test. Detection of resistance genes, erm(A), erm(B), mef(A), tet(M) and tet(O), was done by polymerase chain reaction (PCR). Results: A total of 296 isolates of βHS (240 from north and 21 from south India) were included in the study. Of the 296 βHS, 220 (74%) were GAS, 52 (17.5%) were Group G streptococci and 11 (3.7%), 10 (3.3%) and three (1%) were Group B streptococci, Group C streptococci and Group F streptococci, respectively. A total of 102 (46%) and 174 (79%) isolates were resistant to tetracycline and erythromycin, respectively; a lower resistance to ciprofloxacin (21, 9.5%) was observed. A total of 42 (14%) and 30 (10%) isolates, respectively, were positive for tet(M) and erm(B) genes. Only 13 (5%) isolates were positive for mef(A). None of the isolates were positive for erm(A) and tet(O). There was discordance between the results of E-test and PCR for erythromycin and tetracycline. Interpretation & conclusions: A high level of resistance to erythromycin and tetracycline was seen in βHS in India. Discordance between genotypic and phenotypic results was reported. Absence of erm(A) and tet(O) with high prevalence of tet(M) and erm(B) was observed.