MRSA

MRSA Methicillin-Resistant Staphylococcus aureus Detection and Identification Methods :

Dr.T.V.Rao MD MRSA Methicillin-Resistant Staphylococcus aureus Detection and Identification Methods Dr.T.V.Rao MD 1

Methicillin-resistant Staphylococcus aureus:

Methicillin-resistant Staphylococcus aureus (MRSA) were first reported in the early 1960's and are now regarded as a major hospital acquired pathogen worldwide. The term methicillin resistant is historically used to describe resistance to any of this class of antimicrobials. Methicillin-resistant Staphylococcus aureus Dr.T.V.Rao MD 2

Growing Importance of Staphylococcal Infections in Health care:

Growing Importance of Staphylococcal Infections in Health care Both methicillin sensitive and methicillin resistant Staphylococcus aureus (MSSA, MRSA) can cause invasive and life-threatening infections such as osteomyelitis, septicemia, endocarditis, and pneumonia. Healthcare associated MRSA is of particular clinical importance because it is not only predictably cross resistant to all penicillin's and cephalosporins, but is also typically resistant to multiple other antibiotics. Possibly because of inadequate initial antibiotic treatment, infections with health care associated MRSA result in increased costs and worse outcomes compared to infections with MSSA . Dr.T.V.Rao MD 3

Importance of Surveillance for MRSA:

Studies show that diligent surveillance programs, where patients are screened upon admission, have a tremendous impact in reducing MRSA infection rates. Identifying MRSA carriers, isolating them, and administering antibiotics is an effective method to stop the spread of MRSA Importance of Surveillance for MRSA Dr.T.V.Rao MD 4

MRSA a Important Colonizer:

MRSA may colonize mucosal or epithelial surfaces, the most common colonized site being the anterior nares. Sites of colonization are important not only as a source of subsequent infection, but also serve as a reservoir for transmission. MRSA a Important Colonizer Dr.T.V.Rao MD 5

CDC estimates ……:

Estimates recently published by the Centers for Disease Control (CDC) based on US data put the number of MRSA associated fatalities at more than 18,000/yr. This places MRSA infection fatalities at levels approaching those for the AIDS epidemic in the US. CDC estimates …… Dr.T.V.Rao MD 6

Two types of mrsa categorized as CA-mrsa and ha-mrsa:

Two types of mrsa categorized as CA-mrsa and ha-mrsa The MRSA organism has been sub-categorized as community-acquired MRSA (CA-MRSA) or hospital-acquired MRSA (HA-MRSA) depending upon the circumstances of acquiring the disease. Current data indicate that these represent distinct strains of the bacterial species. MRSA has been found to be especially troublesome in hospital-acquired (nosocomial) infections where patients have open wounds, invasive devices, or weakened immune systems and can be at greater risk for infection than the general public Dr.T.V.Rao MD 7

When you say it is a mrsa:

Resistance occurs when the organism has a mecA gene producing an altered penicillin binding protein, PBP2a (also known as PBP2') and either an oxacillin MIC of 2mg/l or a methicillin MIC of 4mg/l. When you say it is a mrsa Dr.T.V.Rao MD 8

Health care workers too spread MRSA:

In a healthcare setting, MRSA is spread among doctors, patients, nurses, and visitors who come in contact with contaminated surfaces like bed rails or computer keyboards Infected and colonized patients are the reservoir of MRSA both in hospitals and the community with transmission generally being via contact with health workers. Health care workers too spread MRSA Dr.T.V.Rao MD 9

Broth based Tests for Detection of mrsa:

Broth based enrichment media are commonly employed to increase sensitivity. However this is at the expense of speed of result. NaCl is generally added to the base broth along with either methicillin, oxacillin, [and more recently] cefoxitin. Indicator compounds can also be used to give an early indication of the presence of MRSA. Broth based Tests for Detection of mrsa Dr.T.V.Rao MD 10

Solid Agar Media for detection of MRSA:

Solid Agar Media for detection of MRSA Solid Agar Media: There are no universal standardised methods for screening and isolation of MRSA using solid agar media. Many selective media are available, and these rely on inhibitors such as NaCl and/or antibiotics to aid selection, together with a pH indicator to highlight presumptives. Examples are Mannitol Salt Agar containing 7% NaCl with either 4mg/L methicillin or 2mg/L oxacillin Dr.T.V.Rao MD 11

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Cefoxitin replaces the oxacillin disc :

A number of recent studies using cefoxitin disc diffusion method suggest greater reliability than with oxacillin. No special medium or incubation temperature is required and the test is less affected by hyper-producers of penicillinase. Cefoxitin replaces the oxacillin disc Dr.T.V.Rao MD 13

Other screening agar for mrsa detection:

Oxacillin Resistant Screening Agar with 5.5% NaCl and 2mg/L oxacillin; Baird Parker Medium with 8mg/L ciprofloxacin; Mueller Hinton Agar with 4% NaCl and 6mg/L oxacillin. Sensitivity at 24hrs incubation is variable with 48hrs incubation often required for an acceptable result. Other screening agar for mrsa detection Dr.T.V.Rao MD 14

Clsi guidelines to be followed for detection of MRSA:

There is no single method that is suitable for all MRSA strains. Standard methods are published by the British Society for Antimicrobial Chemotherapy (BSAC) and in the USA, by the Clinical Laboratory Standards Institute (CLSI), previously known as NCCLS. Clsi guidelines to be followed for detection of MRSA Dr.T.V.Rao MD 15

Recent Clsi guidelines for Detection of MRSA:

The most recent CLSI supplement (M100-S14) suggests the use of 30µg cefoxitin discs using a breakpoint of <= 19mm as indicative of resistance of S. aureus to oxacillin. Other media based methods include agar, broth based breakpoint methods (2mg/L oxacillin, 4mg/L methicillin) and agar screening methods recommended by CLSI (Approved standard M7-A6). Recent Clsi guidelines for Detection of MRSA Dr.T.V.Rao MD 16

Minimum inhibitory concentration is a reference method:

Minimum Inhibitory Concentration [MIC] by the dilution method has traditionally been the reference method. Minimum inhibitory concentration is a reference method Dr.T.V.Rao MD 17

Bsac recommends ….:

BSAC recommend the use of Mueller Hinton or Columbia agar with 2% NaCl and 10 4 cfu/ml inoculum incubated at 30°C. CLSI recommend Mueller Hinton Agar with 2% NaCl and 10 4 cfu/ml inoculum incubated at 33-35°C . Bsac recommends …. Dr.T.V.Rao MD 18

New chromogenic medium for mrsa detection:

The exact composition of these agars are proprietary, but mostly contain a selective agent in a culture agar (similar to mannitol salt agar with 8 μg /ml cefoxitin) as well as a chomogenic substrate (like X- Phos ) to mark the growing colonies. New chromogenic medium for mrsa detection Dr.T.V.Rao MD 19

chromID S. aureus Specific chromogenic media for Staphylococci:

Direct identification of S. aureus is based on the spontaneous green coloration of α-glucosidase-producing colonies Optimum differentiation of mixed cultures due to the presence of a 2nd substrate. Orientation of identification towards Staphylococci = white, pink or mauve colonies*.Inhibition of other bacteria (Gram + and Gram -) and yeasts. chromID S. aureus Specific chromogenic media for Staphylococci Dr.T.V.Rao MD 20

CHROMagar MRSA(CHROM agar) Microbiology, Paris, France) (CMRSA) were evaluated.:

CHROMagar MRSA (CHROMagar Microbiology, Paris, France) is a new chromogenic medium for the identification of MRSA. The composition of the chromogenic and selective mix is proprietary . The growth of colonies showing any pink or mauve coloration was considered to be positive (indicating MRSA). The procedure was performed as recommended by the manufacturer. CHROMagar MRSA (CHROM agar) Microbiology, Paris, France) (CMRSA) were evaluated . Dr.T.V.Rao MD 21

CHROMagar MRSA(CHROM agar Microbiology, Paris, France) (CMRSA) were evaluated.:

The isolates were inoculated on Columbia agar plates with 5% sheep blood and incubated for 24 h at 35°C. From the resulting cultures, a suspension at a 0.5 McFarland standard was made; subsequently, a swab was dipped in the suspension and streaked on a CHROMagar MRSA plate. The results were read after 24 and 48 h of incubation at 35°C. CHROMagar MRSA (CHROM agar Microbiology, Paris, France) (CMRSA) were evaluated . Dr.T.V.Rao MD 22

BBL™ CHROMagar™ MRSA:

BBL ™ CHROMagar ™ MRSA BBL ™ CHROMagar™ MRSA is a differential medium designed for the direct detection and/or confirmation of MRSA. Selective agents are incorporated for the suppression of gram-negative organisms, most strains of S. epidermidis and yeast. S. aureus hydrolyzes the chromogenic substrate resulting in mauve colonies, thus permitting differentiation from other gram-positive organisms . A cephalosporin is incorporated for the differentiation of methicillin-resistant strains from methicillin-susceptible strains. BBL™ CHROMagar™ MRSA has the potential for detection and identification of MRSA directly from the primary isolation plate. This unique product has the potential to improve turnaround time, utilize less technologist time and allow for greater recovery of MRSA than traditional MRSA screening algorithms. Dr.T.V.Rao MD 23

BBL CHROMagar:

BBL CHROMagar MRSA is a selective and differential medium, which incorporates cefoxitin, for the detection of MRSA from anterior nares specimens. BBL CHROMagar Dr.T.V.Rao MD 24

BBL™ CHROMagar™ MRSA:

BBL™ CHROMagar™ MRSA is a selective and differential medium for the qualitative direct detection of nasal colonization by methicillin-resistant Staphylococcus aureus (MRSA) to aid in the prevention and control of MRSA infections in healthcare settings. The test is performed on anterior nares swab specimens from patients and healthcare workers to screen for MRSA colonization. BBL CHROMagar MRSA is not intended to diagnose MRSA infection nor to guide or monitor treatment for infections. BBL™ CHROMagar ™ MRSA Dr.T.V.Rao MD 25

Biosynth AG (Switzerland) develop a fluorogenic assay for MRSA:

Biosynth AG (Switzerland) develop a fluorogenic assay for MRSA that utilizes the precipitating fluorescent substrate ELF- Phos which produces a bright (green) fluorescent precipitate on selective agar plates at the site of MRSA colony growth. In contrast to fluorescence in solution, solid state fluorescence is rare due to self-quenching in the solid state and most molecules that display solid state fluorescence remain fluorescent in solution as well Biosynth AG (Switzerland) develop a fluorogenic assay for MRSA Dr.T.V.Rao MD 26

Rapid MRSA detection by a latex kit :

A rapid latex agglutination kit available to detect MRSA clinical isolates (MRSA-Screen test Denka Seiken Co Ltd, Tokyo, Japan) based on detecting a specific penicillin binding protein 2a (PBP2a) in comparison to the NCCLS oxacillin salt agar screen plate . Rapid MRSA detection by a latex kit Dr.T.V.Rao MD 27

Need for Molecular Methods in Microbiology:

Need for Molecular Methods in Microbiology The next milestone in the development of diagnostic applications of PCR was the development of real-time PCR assays. These assays use labeled probes that are part of the initial PCR reaction mixture; detection of fluorescence accumulation during thermal cycling is used in place of Southern blots for detection of PCR products within the closed environment reaction tube. Real-time PCR is now the most commonly used method for user-developed assays, although only a few commercial kits incorporate this technology . Dr.T.V.Rao MD 28

Why Automated Methods in MRSA Detection :

Culture-based testing takes days to produce a result—it is too slow to be highly effective in isolating carriers from non-carriers. Enter automated molecular diagnostics that enable hospitals to collect a specimen with a nasal swab and process a test in about an hour. In addition to reducing the spread of MRSA and improving patient outcomes Why Automated Methods in MRSA Detection Dr.T.V.Rao MD 29

Molecular Methods for MRSA Detection :

Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) by PCR can be performed directly from nasal specimens with the IDI-MRSA assay The IDI-MRSA real-time polymerase chain reaction (PCR) assay was designed to provide rapid results from nasal specimens collected in Stuart’s liquid transport medium Molecular Methods for MRSA Detection Dr.T.V.Rao MD 30

IDI-MRSA™:

IDI-MRSA ™ ( Infectio Diagnostic, Inc.), utilizes PCR for the amplification of MRSA DNA and fluorogenic target-specific hybridization probes for the detection of the amplicons . Nasal swab specimens are placed directly in sample preparation buffer, and after additional processing, an aliquot of the specimen is added to PCR reagents containing MRSA specific primers. PCR and detection of MRSA amplicons are performed on the Smart Cycler™ instrument. IDI-MRSA™ Dr.T.V.Rao MD 31

Hand washing still continues to reduce several incidences of MRSA spread in health care:

Hand washing still continues to reduce several incidences of MRSA spread in health care Dr.T.V.Rao MD 32

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