Yamuna Devi Bakthavatchalam

Rapid Screening for Carbapenem Resistant Organisms: Current Results and Future Approaches

Carbapenem producing Enterobacteriaceae (CPE) is a major public health threat. A total of 120 carbapenem resistant E.coli (n=32) and K.pneumoniae (n=88) from blood stream infections were screened for the presence of carbapenem resistant genes KPC, NDM, IMP, VIM, and OXA-48 like using both conventional multiplex PCR and Xpert(®) Carba-R test. Additionally 26 faeces samples were directly screened with Xpert(®) Carba-R test. Of the tested isolates, 40% (n=48) of NDM and 39.2% (n=47/) of OXA-48-like were identified. Co-production of OXA-48 and NDM was seen in 15 (12.5%) isolates. In Xpert(®) Carba-R test, only NDM was identified in 55% (n=66) of tested isolates. Of the tested faeces samples, 12 were identified as carbapenemase producers: nine with NDM, two with the co-production of NDM and VIM and in Klebsiella spp (n=1), NDM and KPC co-production was seen. However, Xpert(®) Carba-R test fails to detect OXA-48 like as compared with multiplex PCR. The sensitivity, specificity, PPV, NPV of Xpert(®) Carba-R test was 100%, 77%, 96% and 100% respectively. Incorporation of OXA-48 like specific sequence in the panel of Xpert(®) Carba-R test may improve its sensitivity and maximize the coverage of assay.

Laboratory detection and clinical implication of oxacillinase-48 like carbapenemase: The hidden threat

Carbapenemase producing Gram-negative pathogen is of great concern for physician. The challenging aspects are treatment option and infection control. Monitoring of respective carbapenemase resistance mechanism is necessary to prevent the outbreaks. Currently, the rapid emergence of oxacillinase (OXA-48) like is alarming. Increasing frequency of OXA-48 is seen than the classical carbapenemase (KPC, NDM, IMP, and VIM) across the world. The blaOXA-48 gene is commonly identified in Escherichia coli and Klebsiella pneumoniae. The transferrable plasmid of OXA-48 is associated with rapid spread and inter-species dissemination. In general, OXA-48-like enzymes weakly hydrolyzes both carbapenem and broad spectrum cephalosporins. Except OXA-163, which effectively hydrolyze cephalosporin. This poor hydrolytic profile obscures the detection of OXA-48-like. It may go undetected in routine diagnosis and complicates the treatment option. Co-production of OXA-48-like with CTX-M-15 and other carbapenemase (NDM, VIM) leads to the emergence of multidrug resistant strains.

Mechanisms of Carbapenem Resistance in K.pneumoniae and E. coli from Bloodstream Infections in India

Introduction: Emergence and global spread of carbapenemase producing Enterobacteriaceae (CPE) are of great concern in healthcare settings. Resistance to carbapenem is mostly conferred by metallo β-lactamase (IMP, VIM and NDM) and carbapenem hydrolyzing class D β-lactamase (OXA-48 like). The aim of this study was to characterise the molecular mechanism of resistance in the clinical isolates of Enterobacteriaceae causing bacteremia and showing resistance to β-lactams, including carbapenems. Materials and Methods: Isolates of E.coli (n=42) and K. pneumoniae (n=134) from blood culture collected during 2013-2015 were screened for carbapenemase production by using carba NP test and the presence of carbapenem resistant genes (KPC, IMP, VIM, NDM and OXA- 48 like). Sequencing was performed for the randomly selected isolates positive for NDM and OXA-48 like.Results: Of the 176 isolates, 97% of the isolates were found to be positive with carba NP test. Carba NP test has the sensitivity, specificity, PPV and NPV of 98%, 50%, 99% and 20% respectively. Each of blaNDM and blaOXA-48 like was seen in 32% of the tested isolates. Co-production of blaNDM and blaOXA48 like and blaVIM and blaOXA48 were seen in 13% and 8% of isolates respectively. Noticeably, 3% of isolates were identified as co-producers of blaNDM, blaVIM and blaOXA48 like. All of the sequenced NDM and OXA-48 like were identified as NDM-1 and OXA-181 variants. Conclusion: Increasing incidence of OXA-48 like is worrisome in developing countries. Because of its weak hydrolytic acivity against broad spectrum cephalosporin and carbapenems, these may go undetected in routine screening. In particular, blaOXA48 like gene is mostly identified on the plasmid and is implicated as the cause for silent spread and outbreaks in hospitalized patients.

Evolving rapid methicillin-resistant Staphylococcus aureus detection: Cover all the bases

The dissemination of methicillin-resistant (MR) Staphylococcus aureus (SA) in community and health-care settings is of great concern and associated with high mortality and morbidity. Rapid detection of MRSA with short turnaround time can minimize the time to initiate appropriate therapy and further promote infection control. Early detection of MRSA directly from clinical samples is complicated by the frequent association of MRSA with methicillin-susceptible SA (MSSA) and coagulase-negative Staphylococcus (CoNS) species. Infection associated with true MRSA or MSSA is differentiated from CoNS, requires target specific primers for the presence of SA and mec A or nuc or fem A gene for confirmation of MR. Recently, livestock-associated MRSA carrying mec C variant complicates the epidemiology of MRSA further. Several commercial rapid molecular kits are available with a different combination of these targets for the detection of MRSA or MSSA. The claimed sensitivity and specificity of the currently available commercial kits is varying, because of the different target combination used for detection of SA and MR.

Molecular characterisation of antimicrobial resistance in Pseudomonas aeruginosa and Acinetobacter baumannii during 2014 and 2015 collected across India

Background: Surveillance of antimicrobial resistance (AMR) is of great importance. Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens and emergence of resistance in these have increased the morbidity and mortality rates. This surveillance study was initiated by the Government of India - Indian Council of Medical Research. The aim of this study is to determine the antimicrobial susceptibility profile and to characterise the enzyme mediated antimicrobial resistance such as extended spectrum beta-lactamases (ESBLs) and carbapenemases among multidrug-resistant (MDR) P. aeruginosa and A. baumannii. Materials and Methods: A multi-centric study was conducted from January 2014 to December 2015 with a total number of 240 MDR P. aeruginosa and 312 MDR A. baumannii isolated from blood, cerebrospinal fluid, respiratory, pus, urine and intra-abdominal infections. Kirby–Bauer disc diffusion was done to determine the antimicrobial susceptibility profile. Further, MDR isolates were characterised by multiplex polymerase chain reaction to determine the resistance genes for ESBLs and carbapenemases. Results: Among the ESBLs, blaVEB (23%), blaTEM (5%) and blaSHV (0.4%) in P. aeruginosa and blaPER (54%), blaTEM (16%) and blaSHV (1%) in A. baumannii were the most prevalent. Likewise, blaVIM (37%), blaNDM (14%), blaGES (8%) and blaIMP (2%) in P. aeruginosa and blaOXA-23like (98%), blaOXA-58like (2%), blaNDM (22%) and blaVIM (3%) in A. baumannii were found to be the most prevalent carbapenemases. blaOXA-51like gene, intrinsic to A. baumannii was present in all the isolates tested. Conclusion: The data shown highlight the wide difference in the molecular mechanisms of AMR profile between P. aeruginosa and A. baumannii. In P. aeruginosa, plasmid-mediated mechanisms are much lesser than the chromosomal mediated mechanisms. In A. baumannii, class D oxacillinases are more common than other mechanisms. Continuous surveillance to monitor the trends in AMR among MDR pathogens is important for implementation of infection control and to guide appropriate empirical antimicrobial therapy.

Diagnosis and management of Panton-Valentine leukocidin toxin associated Staphylococcus aureus infection: an update

The incidence of invasive Staphylococcus aureus (SA) infection has increased in the past decade and is associated with poor outcomes and high mortality rates. Of all the virulence factors, Panton-Valentine Leukocidin (PVL) has received the greatest attention. PVL producing SA strains are more likely to produce severe skin and soft tissue infections (SSTIs) and necrotizing pneumonia. This review focuses on the current evidence on PVL-SA virulence, epidemiology, clinical disease and treatment with relevance to healthcare in India.

Strengths and limitations of various screening methods for carbapenem-resistant Enterobacteriaceae including new method recommended by clinical and laboratory standards institute, 2017: A tertiary care experience

Carbapenemase-mediated carbapenem resistance is a major concern across the world. Rapid detection of carbapenemase-producing organisms is of great importance in clinical settings. However, it is essential to have a test with good sensitivity and specificity. The aim of the study was to compare the performance of RAPIDEC® CARBA NP and modified carbapenem inactivation method (mCIM) recommended by Clinical and Laboratory Standards Institute guideline 2017. A total of ninety carbapenem resistant Escherichia coli and Klebsiella pneumoniae have been tested. The presence of various carbapenemases was screened by conventional multiplex polymerase chain reaction. RAPIDEC® CARBA NP detected 90%, whereas mCIM detected 99% of the study isolates tested. Although RAPIDEC® CARBA NP is a rapid test, the sensitivity is reduced for blaOxa-48Likedetection; while mCIM could pick up blaOxa-48Likeenzymes with excellent sensitivity. Further, organisms producing low carbapenemase activity enzymes, thickness of the inoculum and the disc potency are likely to influence the test results of mCIM with an overnight delay.

Polymyxin Nordmann/Poirel test for rapid detection of polymyxin resistance in Enterobacteriaceae: Indian experience

and Laboratory Standards Institute (CLSI) guidelines (M07– A10)[5] and interpreted as per CLSI guidelines (M100–S26). All the isolates were screened for polymyxin resistance using the rapid polymyxin NP test as previously described.[4] This test reagent contains cation-adjusted Mueller-Hinton broth, phenol red (pH indicator) and 10% anhydrous D(+)-glucose and colistin or polymyxin B at a final concentration of 5 μg/ml. This test involved incubation under aerobic conditions to improve glucose metabolism and accuracy of the results.

Genomic insights of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) with reduced teicoplanin susceptibility: A case of fatal necrotizing fasciitis

OBJECTIVES Glycopeptides are increasingly being used to treat multiresistant methicillin-resistant Staphylococcus aureus (MRSA) infections. Here we report an MRSA isolate with low-level teicoplanin resistance (isolate VB26276) recovered from a patient treated with teicoplanin for fatal necrotizing fasciitis. METHODS Minimum inhibitory concentrations (MICs) of MRSA isolates to vancomycin and teicoplanin were determined by Etest. Reduced glycopeptide susceptibility was screened by Etest GRD (glycopeptide resistance detection) and population analysis profile (PAP) method. Next-generation sequencing (NGS) was also performed to determine the molecular mechanism of antimicrobial resistance and virulence. RESULTS The teicoplanin MIC of MRSA isolate VB26276 was 4μg/mL. NGS showed the presence of mutations in the tcaA and tcaB genes of the tcaRAB operon that determines the level of teicoplanin resistance. In addition, a mutation in the vraR gene was found to be associated with teicoplanin resistance, but not with vancomycin heteroresistance. CONCLUSION Teicoplanin resistance may occur due to point mutations in the teicoplanin resistance operon tcaRAB. Further studies are warranted to determine the contribution of point mutations in tcaRAB to reduced teicoplanin susceptibility.

Typhoid fever: issues in laboratory detection, treatment options & concerns in management in developing countries

Multidrug-resistant Salmonella enterica subsp. enterica serovar Typhi (resistant to ampicillin, chloramphenicol and cotrimoxazole), was significantly reduced with the increased usage of fluoroquinolones and azithromycin. This has led to declining multidrug resistance rates in India with increasing ciprofloxacin nonsusceptibility rates and clinical failures due to azithromycin. However, for the available agents such as ceftriaxone, azithromycin and fluoroquinolones, the dose and duration for treatment is undefined. The ongoing clinical trials for typhoid management are expected to recommend the defined dose and duration for better clinical outcome. We made an attempt to summarize the issues in laboratory detection, treatment options and responses, and the concerns in clinical practice seen in the developing countries.