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HODGE TEST SKILL BASED LEARNING Dr.T.V.Rao MD 1. Role Carbapenems Carbapenems are often used as antibiotics of last resort for treating infections due.

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Presentation on theme: "HODGE TEST SKILL BASED LEARNING Dr.T.V.Rao MD 1. Role Carbapenems Carbapenems are often used as antibiotics of last resort for treating infections due."— Presentation transcript:

1 HODGE TEST SKILL BASED LEARNING Dr.T.V.Rao MD 1

2 Role Carbapenems Carbapenems are often used as antibiotics of last resort for treating infections due to multidrug-resistant gram-negative bacilli, because they are stable even in response to extended spectrum and AmpC -lactamases. However, gram-negative bacilli producing the acquired metallo-lactamases (MBLs) IMP and VIM have been increasingly reported in Asia and Europe Dr.T.V.Rao MD2

3 Emerging Carbapenems Resistance in Gram-Negative Bacilli Significantly limits treatment options for life- threatening infections No new drugs for gram-negative bacilli Emerging resistance mechanisms, carbapenemases are mobile, Detection of carbapenemases and implementation of infection control practices are necessary to limit spread Dr.T.V.Rao MD3

4 Antibiotic Misuse Antibiotic misuse, (sometimes called antibiotic abuse or antibiotic overuse) refers to the misuse and overuse of antibiotics which has serious effects on public health. Antibiotic resistant bacteria is a growing threat and becoming increasingly common. This overuse creates multi- antibiotic resistant life threatening infections by "super bugs, sometimes out of relatively harmless bacteria. Antibiotic abuse also places the patient at unnecessary risk of adverse effects of antibiotics. Dr.T.V.Rao MD4

5 Bugs to Superbugs. Antibiotic resistance develops through gene action or plasmid exchange between bacteria of the same species. If a bacterium carries several resistance genes, it is called multiresistant or, informally, a superbug. Dr.T.V.Rao MD5

6 Carbapenem Resistance: Mechanisms EnterobacteriaceaeCephalosporinase + porin loss Carbapenemase P. aeruginosaPorin loss Up-regulated efflux Carbapenemase Acinetobacter spp.Cephalosporinase + porin loss Carbapenemase

7 Carbapenemases ClassificationEnzymeMost Common Bacteria Class AKPC, SME, IMI, NMC, GES Enterobacteriaceae (rare reports in P. aeruginosa) Class B (metallo- -lactamse) IMP, VIM, GIM, SPM P. aeruginosa Enterobacteriacea Acinetobacter spp. Class DOXAAcinetobacter spp.

8 Klebsiella Pneumoniae Carbapenemase KPC is a class A -lactamase – Confers resistance to all -lactams including extended-spectrum cephalosporins and carbapenems Occurs in Enterobacteriaceae – Most commonly in Klebsiella pneumoniae – Also reported in: K. oxytoca, Citrobacter freundii, Enterobacter spp., Escherichia coli, Salmonella spp., Serratia spp., Also reported in Pseudomonas aeruginosa (Columbia) Dr.T.V.Rao MD8

9 KPCs in Enterobacteriaceae SpeciesComments Klebsiella spp.K. pneumoniae-cause of outbreaks K. oxytoca-sporadic occurrence Enterobacter spp. Sporadic occurrence Escherichia coli Salmonella spp. Citrobacter freundii Serratia spp. Pseudomonas aeruginosa – Columbia & Puerto Rico

10 What Labs Should Do Now Look for isolates of Enterobacteriaceae (especially K. pneumoniae), with carbapenem MIC 2 g/ml or non susceptible to ertapenem by disk diffusion Consider confirmation by Modified Hodge Test Alert clinician and infection control practitioner to possibility of mobile carbapenemase in isolate Dr.T.V.Rao MD10

11 Control of Infections With Carbapenem-Resistant or Carbapenemase-Producing Enterobacteriaceae in Acute Care Facilities A difficulty in detecting CRE is the fact that some strains that harbor bla kpc have minimal inhibitory concentrations (MICs) that are elevated but still within the susceptible range for carbapenems. Because these strains are susceptible to carbapenems, they are not identified as potential clinical or infection control risks using current susceptibility testing guidelines. Dr.T.V.Rao MD11

12 Background to Modified Hodge Test KPCs are class A carbapenemases that reside on transferable plasmids and are capable of inactivating carbapenems, such as imipenem and meropenem. Since carbapenems are often used to treat infections caused by extended-spectrum beta lactamase (ESBL)- producing Gram-negative bacteria, carbapenemase production in Enterobacteriaceae can significantly limit treatment options for life-threatening diseases. KPCs occur most commonly in Klebsiella pneumoniae but have been seen in other species of Enterobacteriaceae as well. Dr.T.V.Rao MD12

13 CLSI – Modified Hodge Test CLSI published a recommendation that carbapenems-susceptible Enterobacteriaceae with elevated MICs or reduced disk diffusion zone sizes be tested for the presence of carbapenemases using the modified Hodge test (MHT). Dr.T.V.Rao MD13

14 Modified Hodge Test The Modified Hodge Test (MHT) detects carbapenemase production in isolates of Enterobacteriaceae. The most common carbapenemase found in Enterobacteriaceae is the Klebsiella pneumoniae carbapenemase (KPC). Other carbapenemase, like the metallo β lactamase (MBL) and the SME-1 in Serratia marcescens, can also produce a positive MHT, but are found infrequently in the United States. Dr.T.V.Rao MD14

15 Purpose of Hodge Test Carbapenemase production is detected by the MHT when the test isolate produces the enzyme and allows growth of a carbapenems susceptible strain (E.coli ATCC 25922) towards a carbapenem disk. The result is a characteristic cloverleaf-like indentation Dr.T.V.Rao MD15

16 Modified Hodge Test The MHT performed on a 100 mm MHA plate. (1) K. pneumoniae ATCC BAA 1705, positive result (2) K. pneumoniaeATCC BAA 1706, negative result; and (3) a clinical isolate, positive result312 Dr.T.V.Rao MD16

17 Reagents and materials Reagents 5 ml Mueller Hinton broth (MHB) or 0.85% physiological saline Mueller Hinton agar (MHA) 10 μg meropenem or ertapenem susceptibility disk E. coli ATCC 25922: 18–24hr subculture Equipment Turbidity meter 35OC ± 2OC ambiant air incubator Supplies Sterile cotton-tipped swabs 1 ml sterile pipette Sterile loop Dr.T.V.Rao MD17

18 Specimen and Quality Control Specimen Test organisms: 18–24 hr subculture Special safety precautions Biosaftey Level 2 Quality control Perform quality control of the carbapenem disks according to CLSI guidelines. Perform quality control with each run. MHT Positive Klebsiella pneumoniae ATCC BAA-1705 MHT Negative Klebsiella pneumoniae ATCC BAA-1706 Dr.T.V.Rao MD18

19 Step 1 and 2 1.Prepare a 0.5 McFarland dilution of the E.coli ATCC in 5 ml of broth or saline. 2Dilute 1:10 by adding 0.5 ml of the 0.5 McFarland to 4.5 ml of MHB or saline Dr.T.V.Rao MD19

20 Step 3 and 4 Streak a lawn of the 1:10 dilution of E.coli ATCC to a Mueller Hinton agar plate and allow to dry 3–5 minutes. Place a 10 μg meropenem or ertapenem susceptibility disk in the center of the test area. Dr.T.V.Rao MD20

21 Step 5 and 6 In a straight line, streak test organism from the edge of the disk to the edge of the plate. Up to four organisms can be tested on the same plate with one drug. Incubate overnight at 35OC ± 2OC in ambient air for 16–24 hour Dr.T.V.Rao MD21

22 Interpretation of MHT After 16–24 hours of incubation, examine the plate for a clover leaf- type indentation at the intersection of the test organism and the E. coli 25922, within the zone of inhibition of the carbapenem susceptibility disk. Dr.T.V.Rao MD22

23 MHT – Positive Test A positive MHT indicates that this isolate is producing a carbapenemase Test has a clover leaf- like indentation of the E.coli growing along the test organism growth streak within the disk diffusion zone. Dr.T.V.Rao MD23

24 Showing the Results on HMT testing A positive test indicates carbapenemase production by the test microorganism. By producing carbapenemase, the test microorganism is able to inactivate the carbapenem that diffuses from the disk after the disk has been placed on the Mueller Hinton Agar. This allows carbapenem susceptible E. coli ATCC® 25922* to grow toward the disk Dr.T.V.Rao MD24

25 MHT – Negative Test A negative MHT indicates that this isolate is not producing a carbapenemase Test has no growth of the E.coli along the test organism growth streak within the disc diffusion. Dr.T.V.Rao MD25

26 Showing Positive and Negative isolates by HMT Isolates A and C are negative for KPC. Isolates B and D are positive for KPC as indicated by arcing growth of the carbapenem-sensitive E coli along the clinical KPC isolates toward the carbapenem disk. Dr.T.V.Rao MD26

27 What Acquired metallolactamases do (MBL) The MBLs efficiently hydrolyze all -lactams, except for aztreonam, in vitro. Therefore, detection of MBL- producing gram- negative bacilli is crucial for the optimal treatment of patients and to control the spread of resistance Dr.T.V.Rao MD27

28 Lee et al - reports Carbapenemase detection methods Lee et al. have reported that the Hodge test can be used to screen carbapenemase- producing gram-negative bacilli and that the imipenem (IPM)-EDTA double-disk synergy test (DDST) can distinguish MBL-producing from MBL-nonproducing gram- negative bacilli Dr.T.V.Rao MD28

29 Phenotypic detection with Hodge test a Minimal requirement Carbapenem resistance and carbapenemase production conferred by blaNDM-1 is detected reliably with phenotypic testing methods currently recommended by the Clinical and Laboratory Standards Institute, including disk diffusion testing and the modified Hodge test Dr.T.V.Rao MD29

30 Modified Hodge Test Lawn of E. coli ATCC :10 dilution of a 0.5 McFarland suspension Imipenem disk Test isolates Described by Lee et al. CMI, 7, Dr.T.V.Rao MD30

31 Modified Hodge Test Preliminary results suggest that any of the three carbapenem disks work in the Modified Hodge Test Dr.T.V.Rao MD31

32 What Labs Should Do Now Look for isolates of Enterobacteriaceae (especially K. pneumoniae), with carbapenem MIC 2 g/ml or non susceptible to ertapenem by disk diffusion Consider confirmation by Modified Hodge Test Can submit initial isolate to CDC via NJ State Lab for confirmation by bla KPC PCR if KPC-producers not previously identified in hospitals isolate population Alert clinician and infection control practitioner to possibility of mobile carbapenemase in isolate Dr.T.V.Rao MD32

33 Are we losing the Value of Carbapenems Carbapenems are the only antibiotics reliably active against many otherwise multi-resistant gram- negative opportunist bacteria, particularly those with extended-spectrum beta-lactamases (ESBLs) The growing emergence and diversity of carbapenemase producing strains is therefore a major concern. Dr.T.V.Rao MD33

34 CDC reports the new genetic mechanisms The isolate, Klebseilla pneumoniae , was shown to possess a metallo-beta-lactamase (MBL) but was negative for previously known MBL genes. Gene libraries and amplification of class 1 integrons revealed three resistance- conferring regions; the first contained bla(CMY-4) flanked by ISEcP1 and blc. The second region of 4.8 kb contained a complex class 1 integron with the gene cassettes arr-2, a new erythromycin esterase gene; ereC; aadA1; and cmlA7 Dr.T.V.Rao MD34

35 How we can Improve Hodge Test The Hodge test is a simple method for screening MBL-producing isolates, but occasional isolates show false-negative results. The test can be improved by using an IPM disk to which 10 l of 50 mM zinc sulfate (140 g/disk) has been added (Table 3) or by using Mueller-Hinton agar to which zinc sulfate has been added to a final concentration of 70 g/m Dr.T.V.Rao MD35

36 Genetic origin of the NDM-1 An intact ISCR1 element was shown to be downstream from the qac/sul genes. The third region consisted of a new MBL gene, designated bla(NDM-1), flanked on one side by K. pneumoniae DNA and a truncated IS26 element on its other side. The last two regions lie adjacent to one another, and all three regions are found on a 180-kb region that is easily transferable to recipient strains and that confers resistance to all antibiotics except fluoroquinolones and colistin. NDM- 1 shares very little identity with other MBLs, with the most similar MBLs being VIM-1/VIM-2, with which it has only 32.4% identity. Dr.T.V.Rao MD36

37 Molecular configuration of NDM-1 NDM-1 also has an additional insert between positions 162 and 166 not present in other MBLs. NDM-1 has a molecular mass of 28 kDa, is monomeric, and can hydrolyze all beta-lactams except aztreonam. Compared to VIM-2, NDM-1 displays tighter binding to most Cephalosporins. Dr.T.V.Rao MD37

38 NDM genetic coding differs from other recent isolates Compared to VIM-2, NDM-1 displays tighter binding to most cephalosporins, in particular, cefuroxime, cefotaxime, and cephalothin (cefalotin), and also to the penicillins. NDM-1 does not bind to the carbapenems as tightly as IMP-1 or VIM-2 and turns over the carbapenems at a rate similar to that of VIM-2. In addition to K. pneumoniae , bla(NDM-1) was found on a 140-kb plasmid in an Escherichia coli strain isolated from the patient's feces, inferring the possibility of in vivo conjugation Dr.T.V.Rao MD38

39 Phenotypic detection with Hodge test a Minimal requirement Carbapenem resistance and carbapenemase production conferred by blaNDM-1 is detected reliably with phenotypic testing methods currently recommended by the Clinical and Laboratory Standards Institute, including disk diffusion testing and the modified Hodge test Dr.T.V.Rao MD39

40 Follow me for More Articles of Interest on Infectious Diseases Dr.T.V.Rao MD40

41 Created by Dr.T.V.Rao MD for e learning resources for Medical Microbiologists in Developing Countries Dr.T.V.Rao MD41


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