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

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 MD

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 MD

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 MD

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 MD

Carbapenem Resistance: Mechanisms Enterobacteriaceae Cephalosporinase + porin loss Carbapenemase P. aeruginosa Porin loss Up-regulated efflux Acinetobacter spp.

Carbapenemases Classification Enzyme Most Common Bacteria Class A KPC, SME, IMI, NMC, GES Enterobacteriaceae (rare reports in P. aeruginosa) Class B (metallo-b-lactamse) IMP, VIM, GIM, SPM P. aeruginosa Enterobacteriacea Acinetobacter spp. Class D OXA

Klebsiella Pneumoniae Carbapenemase KPC is a class A b-lactamase Confers resistance to all b-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 MD

KPC’s in Enterobacteriaceae Species Comments 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

What Labs Should Do Now Look for isolates of Enterobacteriaceae (especially K. pneumoniae), with carbapenem MIC ≥ 2 mg/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 MD

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 blakpc 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 MD

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 MD

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 MD

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 MD

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 MD

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 MD

Reagents and materials 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 MD

Specimen and Quality Control 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 MD

Step 1 and 2 1.Prepare a 0.5 McFarland dilution of the E.coli ATCC 25922 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 MD

Step 3 and 4 Streak a lawn of the 1:10 dilution of E.coli ATCC 25922 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 MD

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 MD

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 MD

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 25922 growing along the test organism growth streak within the disk diffusion zone. Dr.T.V.Rao MD

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 MD

MHT – Negative Test A negative MHT indicates that this isolate is not producing a carbapenemase Test has no growth of the E.coli 25922 along the test organism growth streak within the disc diffusion. Dr.T.V.Rao MD

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 MD

What Acquired metallo—lactamases 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 MD

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 MD

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 MD

Modified Hodge Test Test isolates Imipenem disk Lawn of E. coli ATCC 25922 1:10 dilution of a 0.5 McFarland suspension Test isolates Imipenem disk Described by Lee et al. CMI, 7, 88-102. 2001. Dr.T.V.Rao MD

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

What Labs Should Do Now Look for isolates of Enterobacteriaceae (especially K. pneumoniae), with carbapenem MIC ≥ 2 mg/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 blaKPC PCR if KPC-producers not previously identified in hospital’s isolate population Alert clinician and infection control practitioner to possibility of mobile carbapenemase in isolate Dr.T.V.Rao MD

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 MD

CDC reports the new genetic mechanisms The isolate, Klebseilla pneumoniae 05-506, 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 MD

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 MD

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 MD

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 MD

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 05-506, 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 MD

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 MD

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