Staphylococcus aureus

Staphylococcus aureus

1.0 Introduction

1.1 Staphylococcus aureus

Staphylococci (genus Staphylococcus),are round, gram positive cocci which can divide in any plane and tend to be seen as distinctive grapelike clusters upon gram staining (Figure1). They are non motile; non flagellate and non spore forming. Staphylococci are facultative anaerobic but grow best aerobically. The ability of Staphylococcus aureus (S.aureus) to produce coagulase distinguishes it from other Staphylococcus strains. (Ryan 1994) (Nahimana, Francoli & Blanc 2006)Although S. aureus is part of the normal flora of healthy individuals and is carried in the anterior nares of approximately 20-30% of humans(Meehan et al. 2009)-refer toTable1, strains such as MRSA have the potential to cause disease. (Lelievere, Lina & Jones 1999) (Currie et al. 2008)

Table 1 Features of human Staphylococci

SPECIES

COAGULASE

COMMON HABITAT

CATHETER COLONIZATION

EXOTOXIN PRODUCTION

S. aureus

+

Anterior nares, perinium

+

+

S.albus

-

Anterior nares, skin

+

-

S.saphrophyticus

-

Urinary tract

+

-

1.2 Emergence of MRSA

When penicillin first came into use, many S.aureus strains were susceptible to its affects. However, now, mainly due to its over prescription as an antimicrobic S.aureus emerged as resistant over time due to penicillinase production of some strains.

1.3MRSA- Mechanisms of bacterial resistance

Resistance tends to be acquired by transduction or via conjugation between strains of S.aureus. Some multi-resistant strains have developed resistance to penicillin's and cephalosporin's and therefore led to epidemics in community and especially nosocomial infections almost globally (Chambers & De Leo September 2009) These strains are commonly referred to as methicillin resistant S.aureus, though they are resistant to many beta-lactam antibiotics. (Gold 1996) (Sevin, Larmaraud-Sevin & Legrand 1999)The main mechanism by which MRSA resistance is achieved is down to chromosomal attainment of the mec A gene. This gene codes for a new peptide - penicillin binding peptide PBP-2 - that has low affinity for β-lactams. The PBP-2 peptide is capable of replacing other staphylococci transpeptidases in cell wall synthesis in the presence of β-lactams, thereby establishing resistance. (Chambers & De Leo September 2009) Figures2 and 3 appendices

'Hospitals inGalway,DublinandCorkhad the highest number of MRSA infections last year despite an overall fall in patients contracting the superbug'. (O'Regan 2009, Tuesday July 14) (O'Regan Tuesday October 13 2009)

‘Over 7,500 patients were surveyed in 44 acute Irish hospitals with the overall figure of MRSA infection emerging as under 0.5% -Irish hospitals recorded an overall figure of 4.9%'(Health Service Executive, Ireland (HSE) 2007)

A recent study identified 12% of MRSA isolates as being community-associated, and skin and soft tissue infections were more common among community-associated cases compared with those acquired in hospital or in healthcare associated institutions (Naimi TS Le Dell KH 2003)

1.3 The principle behind Chromogenic agars

Since the first report of methicillin-resistant Staphylococcus aureus (MRSA) as a major nosocomial pathogen in the 1960s,the incidence of infections due to this organism has continued to rise (Gold 1996) (Klevens October 17, 2007). Infections caused by MRSA increase the length of hospital stays and are responsible for rising health care costs, and have a high mortality rate (Carbon 1999) (Jonathan R. Edwards 2009)

In the screening of patients who are at risk to MRSA infection, chromogenic medium, selective for MRSA must be sensitive and capable of detecting suspect colonies efficiently. These agars usually consist of many elements that allow for the growth of only the desired microorganism, i.e. MRSA. These selective mediums inhibit most other bacterial growth and where growth is not inhibited, colonies that form will yield a different colour or no colour at all to allow for the differentiation. (Ryan 1994)

1.4 The chromogenic media used for the detection of MRSA isolates

1.4.1 BIOMÉRIEUX chromID™ MRSA Agar

This is a clear medium that bases the isolation of MRSA upon both its resistance to cefoxitin, which is included in the media and chromagen that detects the α-glucosidase activity, which gives rise to the distinctive green colonies. Inhibition of other bacterial growth and the differentiation between MRSA and other Staphylococcal species; that can yield white; pink and/or mauve colonies leads to the increased reliability; sensitivity and specificity of this medium in the screening of MRSA.(Biomerieux 2009)

1.4.2 OXOID BRILLIANCE™ MRSA CHROMAGAR

This is an opaque medium that consists of chromagen that yields denim blue colonies due to the phosphatase activity of many Staphylococci species. To enable differentiation between these strains and MRSA, a marker (other than cefoxitin) is also included, which suppresses the phosphatase enzyme of all other Staphylococci apart from MRSA.This then allows for the isolation of only MRSA present to produce denim blue colonies thereby, increasing the sensitivity and specificity of the media for the detection of MRSA. (Oxoid Ltd 2008)

1.4 Objectives

The aim of this study was to evaluate the ability of a new MRSA chromogenic agar; Oxoid Brilliance MRSA agar, for the detection of MRSA in clinical swabs post 18h incubation compared to that of the agar currently in use; bioMerieux chromID MRSA agar, after 18h and a further 24h re-incubation, at the request of the Microbiology Department at GUH. bioMerieux chromID™ MRSA agar and Oxoid Brilliance™ agar are selective and differential chromogenic mediums for the qualitative detection of methicillin resistant Staphylococcus aureus (MRSA) colonization. The test was performed using swab specimens to screen for MRSA colonization. Monitoring and detection of MRSA infections in this manner may aid in the surveillance and reduction of MRSA outbreaks. (Perry, Rennison & Butterworth Oct. 2004), ( (Nahimana, Francoli & Blanc 2006)(Muto, Jernigan & Ostrowsky 2003)(Sevin, Larmaraud-Sevin & Legrand 1999)

The comparison was based upon their sensitivity; specificity; ease of use; cost effectiveness and rapidity in obtaining reliable confirmatory results

2.0 Materials and Methods

2.1 Materials used

2.1.1 Media used

  • BioMerieux MRSA ID chromagar plates
  • Oxoid Brilliance MRSA chromagar plates
  • CLED agar plates
  • Muller Hinton agar plates

2.1.2 Antimicrobial Susceptibility Testing (AST)

The materials used were;

  • Cefoxitin discs (30µg) (Oxoid)
  • Oxoid Disc Dispenser (Cefoxitin 30)
  • Vortex genie 2™ (BioMerieux)
  • Vitek densiChek™ (BioMerieux)
  • ATCC control strains
  • Saline (5ml sterile tubes )

2.1.3 REAGENTS used

  • Pastorex™ Staph-plus latex reagent kit
  • Sterile swabs
  • Sterile 1µl loops
  • Plate racks
  • Test-tube racks
  • 4°C cold room
  • 36°C +/- 1°C O² incubator
  • BioMerieux ID-ASE™ Catalase reagent
  • MRSA clinical specimen swabs (received daily)- in charcoal transport medium

2.2 Methods of screening for MRSA

2.2.1 Specimen collection and transport

The sterile swab is the most convenient method of specimen collection for MRSA screening.. (Ryan 1994), therefore the use of swabs within transport tubes containing charcoal was ideal, since they are designed to maintain the viability of any bacteria present without significant increase in growth due to being non nutritive. (Aimes 1967)

2.2.2 Specimens used

A total of 325 swabs were used in this trial-sourced from GP and hospital patients (December 2009-January 2010). The most common swabs used for MRSA screens tended to be:

  • Nasal
  • Axilla
  • Groin and
  • Body

Table 4.1 Sensitivities of Body Sites for the Detection of MRSA- Results

Graph4.2 Percentage of MRSA isolation based on swab site- Results

‘Active surveillance for patients colonized with methicillin resistant Staphylococcus aureus (MRSA) is recommended to prevent MRSA infections in health care settings. The sensitivity of patient screening for MRSA colonization is partially dependent on the body sites sampled. The nose and open skin areas (i.e., wounds and device exit sites) are considered the most important sites for colonization However, studies have suggested that a substantial proportion of colonized patients will be missed if only these anatomic sites are sampled' (Journal of Clinical Microbiology 2008)

2.2.3 Good Laboratory Practice

All specimens received were considered as infectious.

Asceptic technique and normal precautions were observed while handling all specimens, reagents and microbial cultures throughout the evaluation.

2.3 Culture methods

2.3.1Direct Inoculation

Each swab was directly inoculated onto both MRSA selective agars; streaking for discrete colonies was not required (Figure 4-appendices). The sequence of inoculation was randomized to reduce bias (Haekel, Rainer and Wosniok, Werner, 2007).

Day 1: BioMerieux MRSA agar was swabbed first then Oxoid Brilliance MRSA agar.

Day 2: Oxoid Brilliance MRSA agar was swabbed first then BioMerieux MRSA agar. The alternation of inoculation continued as such for each day of the trial, until all Oxoid plates were used.

Plates were inoculated directly on the day of receipt of the swab¹, incubated at 36°C aerobically, and read after 20 and 48 h (18-20 h incubation recommended for Oxoid media. (Oxoid Ltd 2008)

¹Prior to media inoculation, all plates used were allowed to reach room temperature. Any unused plates were re-fridgerated (4°C).

2.4 Identification and confirmation of MRSA isolates

Colonies suggestive of MRSA were confirmed asStaphylococcus aureusby the use of latex agglutination kits and antibiotic susceptibility testing (AST) for MRSA confirmation.

The plates were read as per manufacturers' recommendations:

BioMerieux plates post 20 and 48 hours aerobic incubation (36+/- 1°C)

· Oxoid plates post 20 hours aerobic incubation (36+/- 1°C)

Purity plates were set up on CLED agarwhere low level colonization occurred (<10 colonies) and/or if growth was mixed.

2.4.1 Latex Agglutination

Staph Latex - PASTOREX ™ - BIO-RAD - Test principle

The test is designed to allow for the differentiation of S.aureus from coagulase negative Staphylococci(CNS). The combination of fibrinogen, IgG and anti-capsular monoclonal antibodies and protein A (‘clumping factor')allows for the recognition of S.aureus bythe agglutination which forms upon emulsifying a few suspect colonies with the reagent mixture. This agglutination is visible macroscopically within a few seconds approximately. See Diagram 1.

A positive result was visible agglutination with the latex reagent.

No agglutination occurred with the negative control thus ensuring results were valid

(BIO-RAD 2010)

2.4.2Antimicrobial Susceptibility Testing

2.4.2.1Cefoxitin Disk Diffusion

Distinguishing presumptive MRSA isolates was an essential part in determining whether true MRSA's were isolated.

For MRSA, the CLSI recommend the use of 30µg cefoxitin disk on Mûller Hinton agar with 0.5 Mc Farland suspension inoculums, using sterile swabs. (Swenson 2005) (Clinical and Laboratory Standards Institute 2003)After incubation for 24h, the zone diameters were measured in mm, and compared to published figures for interpretation. Zone sizes ≤21mm confirmed MRSA isolation (table 4). (Clinical and Laboratory Standards Institute 2005)

All colonies showing resistance to Cefoxitin were re-tested for the presence of clumping factor. (Colonies tested were taken from directly around the zone diameter).

The Clinical and Laboratory Standards Institute (CLSI) recommends the Cefoxitin disk screen test, the latex agglutination test for PBP2a, alternative methods of testing for MRSA (Clinical and Laboratory Standards Institute 2005)

Table 2 CLSI published figures of zone diameter limitation in the confirmation of MRSA

Susceptible implies that the organism is readily inhibited by the antimicrobic

Resistant implies that the organism is not inhibited by the antimicrobic

2.5Quality Control.

As a validation procedure, Staphylococcus aureus ATCC® 43300 MRSA positive and Staphylococcus aureus ATCC® 25923 MRSA negative were used as daily Internal quality control (IQC) strains- refer to Table3a. These were the only ATCC strains available for use at the time of this trial; see Tables 3b and 3c for both manufacturers' recommendations.

Table 3a IQC STRAINS EXPECTED RESULTS

STRAIN

RESULTS

OXOID

BIOMÉRIEUX

S. aureus

ATCC 43300

Blue colonies

Green colonies

S. aureus

ATCC 25923

Recommended ATCC strains for use with bioMerieuxchromID™ MRSA agar

S. aureus ATCC® 29213 - mecAnegative Expected Result: Inhibition

S. aureus ATCC® 25923 - mecAnegative Expected Result: Inhibition

S. aureus ATCC® 43300 - mecA positive Expected Results : Green colonies

Table 3b (Biomerieux 2009)

Recommended ATCC strains for use with Oxoid Brilliance™ MRSA agar

Staphylococcus aureus NCTC10442 Expected results: Blue colonies

Staphylococcus aureus ATCC®33591 Expected results: Blue colonies

Staphylococcus epidermidis ATCC®12228 Expected results: Inhibited

Staphylococcus aureus ATCC®25923 Expected results: Inhibited

Table 3c (Oxoid Ltd. 2008)

3.0 How should Clinical Laboratories screen for MRSA?

Laboratory screening for MRSA lends itself to being a complex balance between;

  • Sensitivity
  • Specificity
  • Cost
  • Speed of reporting results

Both Oxoid Brilliance™ and bioMerieux chromID™,are chromogenic media that combine primary growth factors among others, which claim to enhance their ability and sensitivity to differentiate MRSA from CNS.

3.1The chromogenic media used in the identification of MRSA isolates

Oxoid™ brilliance MRSA chromagar

  • MRSAappeared as denim blue / navy colonies on an opaque medium
  • Non MRSA appeared as either no growth (inhibition) or white colonies

bioMêriux™ MRSA ID chromagar

  • MRSA appeared as green colonies on a clear medium
  • Non MRSA appeared as either no growth (inhibition) or colourless colonies

3.2 Media Interpretation

Image 1 Actual images of MRSA ATCC 43300 on both media

Image taken 08 February 2010

4.0 Results

4.1 Result reporting protocol

For the purpose of this study the result reporting protocol was as follows:

If presumptive MRSA (PMRSA) colonies grew on a medium and were later confirmed as MRSA (CMRSA) - the result was considered a true positive.

When a sample was found to be negative for MRSA, on both media, the result was assumed as a true negative.

If MRSA - like colonies were yielded, but were not confirmed via further testing, the result was deemed to be a false positive.

An isolate that was confirmed as MRSA on one media, but failed to grow on the other, was resulted as a false negative.

If either medium failed to allow growth of MRSA when the other medium did, this was resulted as a false negative.

4.2 Results obtained

The results obtained for both media are summarized below and in Table 4.1 for

bioMerieux chromID and Oxoid Brilliance respectively. (n=336)

Test

media

bioMerieux chromID

MRSA

xoid Brilliance

MRSA

Absent

Present

Totals

Absent

Present

Totals

Test Positive

3

43

46

9

27

36

Test Negative

290

0

290

287

13

300

Totals

293

43

336

296

40

336

4.3 Data analysis

From the results obtained, (Table 4.1), the respective sensitivities; specificities; positive and negative predictive values (PPV AND NPV) for both media were calculated as follows;

The SPECIFICITY of a test is the probability that a test will produce a true negative result when used on a non-infected person/specimen.

TN/TN+FP

287/287+9 = 96.9%Oxoid

290/290+3 = 98.9%BioMerieux

TheSENSITIVITYof a test is the probability that it will produce a true positive result when used on an infected person/specimen

TP/TP+FN

27/27+13 = 67.5%Oxoid

43/43+0 =100%BioMerieux

ThePOSITIVE PREDICTIVE VALUEof a test is the probability that a person is infected when a positive test result is observed.

TP/TP+FP

27/27+9 = 75%Oxoid

43/43+3 = 93.4%BioMerieux

TP - true positive FP - false positive TN - true negative FN - false negative

These calculated figures are summarized overleaf as Table 4.2 and by Graph 4.3

Table 4.2 Results for both media evaluated in relation to their sensitivity; specificity; positive and negative predictive values (PPV/NPV)

SENSITIVITY

SPECIFICITY

PPV

NPV

OXOID

67.5%

96.9%

75%

95.6%

BIOMÉRIEUX

100%

98.9%

93.4%

100%

Graph 4.3

Graphical representation of results for both media evaluated

It can be seen from these results both media have similar specificities for MRSA, with bioMerieux slightly outperforming Oxoid in this area. However, chromID showed a substantially better performance with regards to sensitivity. Even after 20h incubation, a detection rate of approximately 90% for MRSA, isolated as green colonies on chromID™ was observed, this increased to 100% post 48h incubation, giving a sensitivity of 100% for bioMerieux chromId™. In Comparison, a 62.7% detection rate and 67.5% calculated sensitivity was resulted for Oxoid Brilliance™. This is in contrast to previously reported figures of 99.2% (Oxoid Ltd. 2008) for sensitivity of this medium. bioMerieux chromId™ proved far superior in regards to its sensitivity for the detection of MRSA.

4.4 Distribution of MRSA isolates based on specimen swab site

The main human habitat of MRSA tends to be the anterior nares and skin. These results demonstrate again bioMerieux chromID as being more sensitive at detecting MRSA colonization than Oxoid Brilliance due to its ability to not only detect MRSA from sites that are predisposed to increased MRSA presence, but also of those sites that may contain a low level of colonization. (Graph 4.4 ; 4.5 &Table 4.5)

Graph 4.4Percentage of MRSA isolates based on specimen swab site type (n=43)

Nasal

Body

Other

Total

131

77

117

% Specimens Received

40.3

23.7

36

True Positives

12

7

8

% MRSA Carriage

44

20.7

30

Table 4.5

Sensitivities of Body Sites for the Detection of MRSA among Colonized Patients Identified By Culture Based Screening during the Comparison Study (December 2009-January 2010) (Other swab sites included - Ankle; wound; groin; ulcer and catheter site).

Graph 4.5 percentage yield of MRSA on both media

Specimen ID

Zone diameter to cefoxitin(mm)

Latex reaction

21/12/2009

A67

RESISTANT (14)

POSITIVE

A75

RESISTANT (14)

POSITIVE

A81

RESISTANT (12)

POSITIVE

A75

RESISTANT (12)

POSITIVE

A67

RESISTANT (6)

POSITIVE

04/01/2010

G14

6MM RESISTANT (6)

POSITIVE

G85

11MM RESISTANT (11)

POSITIVE

G05

13MM RESISTANT (13)

POSITIVE

G79

6MM RESISTANT (6)

POSITIVE

06/01/2010

K57

RESISTANT (12)

POSITIVE

K68

RESISTANT (6)

POSITIVE

K72

RESISTANT (12)

POSITIVE

K76

RESISTANT (10)

POSITIVE

K82

RESISTANT (16)

POSITIVE

K84

RESISTANT (8)

POSITIVE

K55

RESISTANT (15)

POSITIVE

K69

RESISTANT (6)

POSITIVE

K56

RESISTANT (12)

POSITIVE

4.5 Results for AST

Table 4.6 Confirmation of MRSA isolates on Oxoid Brilliance™

Where low level or mixed growth occurred of PMRSA colonies, AST was performed to confirm each isolate as MRSA

4.5.2 AST results for false negatives that grew on Oxoid Brilliance™

Table 4.7 Recheck of false negatives on Oxoid Brilliance ™ which were true positives on bioMeriux chromID™.

Specimen ID

Growth observerd

Swab site

Latex reaction

K84

WHITE COLONIES

BODY

POSITIVE

K05

NO GROWTH

NASAL

POSITIVE

K52

WHITE COLONIES

BODY

POSITIVE

K66

BLUE COLONIES

AXILLA

POSITIVE

K69

>10 BLUE COLONIES

BODY

POSITIVE

A number of false negatives observed for Oxoid Brilliance yielded growth of white; pink and mauve colonies. These were latex and AST tested, and subsequently confirmed as MRSA (refer to Table 4.7). This suggests that Oxoids medium is either not sensitive enough to the phosphatase activity of MRSA or that there is a delay in its production by the microorganism. Either way, this only reinforces the inadequacies of the medium for MRSA detection.

K66 AND 69 only became positive (blue colonies) at greater than 48h incubation.Plates were inoculated - 04/01/2010, and did not yield blue colonies until - 06/01/2010

All the above were positive on bioMerieux chromID™ (48h incubation)

4.6 ATCC controls tested on both media

Table 4.8 Resultsfor ATCC strains tested on both media

Control strain

Oxoid Brilliance™

bioMerieux chromID™

Staph.luteus

NO GROWTH

NO GROWTH

Staph. aureus MRSA negative ATCC 29213

GROWTH LTX +

NO GROWTH

Staph.aureus MRSA positive ATCC 43300

GROWTH BLUE LTX +

GROWTH GREEN LTX +

Staph. aureus MRSA neg. ATCC 976

GROWTH LTX -

NO GROWTH

Staph. sciuri ATCC 29061

GROWTH LTX +

NO GROWTH

Staph. saprophyticus ATCC 15305

NO GROWTH

NO GROWTH

Staph.epidermidis ATCC 12228

NO GROWTH

NO GROWTH

Staph. aureus

NO GROWTH

NO GROWTH

Staph. aureus ATCC 977

GROWTH LTX +

NO GROWTH

Enterococcus faecalis ATCC 51299 VRE positive

GROWTH LTX -

NO GROWTH

Enterococcus faecalis ATCC 29212 VRE negative

GROWTH LTX -

GROWTH LTX -

See Image 2

Growth indicates denim blue / green colonies grew on the agar. (Oxoid/Biomerieux respectively).

No growth indicates either inhibition of growth or growth of non - PMRSA colonies(Latex negative CN

The lack of specificity by Oxoid Brilliance™ MRSA agar was evident here by the growth of numerous ATCC control strains that were non- MRSA. Each non- MRSA strains that grew on Oxoids medium yielded blue colonies, identical to that expected for MRSA isolates. bioMerieux chromId™ MRSA agar, did allow the growth of one non-MRSA, ATCC control strain, however colonial morphology and colour differed from that expected of an MRSA isolate, thus allowing for differentiation between significant and non significant growth.(see Image 2 ) This reduced the need for unnecessary further testing, thereby reducing costs and increased rapidity of reporting results.

Growth on both media of Enterococci -VRE negative ATCC 29212 strain

The results obtained for sensitivity and specificity for both Oxoid Brilliance™ and bioMerieuxchromID™ were used to assess their theoretical predictive values (probability for TP; FP; FN and TN),

4.7 Hypothetical assessment of both media for routine use

Table 4.8Estimated predictive values for both media with respect to MRSA detection

bbioMerieuxchromID™

MRSA PREVALANCE

49%

Sensitivity

100%

Specificity

98.%

probability that it will be positive

5.85%

probability that it will be negative

94.1%

For any positive test result:

probability that it is a true positive
["positive predictive value"]PPV

83.7%

probability that it is a false positive

16.25%

For any negative test result:

probability that it is a true negative
["negative predictive value"]NPV

100%

probability that it is a false negative

0

Oxoid Brilliance™Bottom of Form

MRSA PREVALANCE

49%

Sensitivity

67.5%

Specificity

96.9%

probability that it will be positive

6.25%

probability that it will be negative

93.74%

For any positive test result:

probability that it is a true positive
["positive predictive value"]PPV

52.87%

probability that it is a false positive

47.13%

For any negative test result:

probability that it is a true negative
["negative predictive value"]NPV

98.30%

probability that it is a false negative

1.69%

Using the results obtained for sensitivity and specificity for both media evaluated, the theoretical predictive values were estimated .The results in Table 4.8 were estimated, given that the prevalence of MRSA within the Republic of Ireland is 0.049 (4.9%), (Health Service Executive, Ireland (HSE) 2007)

The estimated results above demonstrate that should Oxoid Brilliance™ agar, replace bioMerieux chromID, as the routine method of culture screening for MRSA, the detection rate would be significantly reduced (83.7% versus 52.8% for bioMerieux and Oxoid respectively- see above Table 4.8).

This would also lead to a drop in treatment administration and ultimately an increase in HA-MRSA and CA-MRSA infections.

5.0 Discussion

MRSA is a serious public health problem which exerts huge pressure on the healthcare system. A strategy used to combat this is admission screening using several MRSA swabs taken from mucocutaneous colonization sites of high-risk patients (MRSA screening). It has also been reported that the speed with which MRSA carriage is detected has an important role to play as a key component of any effective scheme to prevent the pathogen from spreading. (Sturenburg & LADR GmbH 2009) Rapid reporting of screening results will improve MRSA control, provided that a clear action plan for positive cases is in place and is being followed. (SARI infection control subcommittee 2008)

An effective culture screening method is direct inoculation of specimen swabs on a well-performing MRSA-selective chromogenic agar. This method usually produces a positive result after 24 h of incubation in >95% of true-positive cases (Diederen 2006) and is sufficient for most initial treatment and infection control decisions. Full antimicrobial susceptibilities, by the Cefoxitin disk diffusion method, can then be performed the next day (48h agar)to confirm MRSA isolation. (Bannerman 2003)

Since current MRSA culturing methods involve a 2-3 day delay before the final results are available, rapid culturing media, such as Oxoid Brilliance™ MRSA agar, have been developed and should they prove reliable, their implementation would reduce the time of detection of MRSA carriers from 48-72 to 18-20.

This study took into account all aspects of an effective media for the detection of MRSA from clinical specimen swabs, basing the results obtained upon sensitivity; specificity; PPV; NPV and economic viability to determine the best media that would suit this purpose.

5.1 Sensitivity

Post 24h incubation, nasal positive specimens yielded green colonies on bioMerieux chromID™ agar, most swabs from other sites became positive after 48h incubation - this was most likely due to the increased presence of MRSA in anterior nares than other body sites, thus, they require further incubation to produce significant growth for detection-, therefore, the 48h incubation with BioMerieux plates seems to be justified. Although Oxoid Brilliance™ MRSA media does not require further incubation, their plates frequently showed MRSA isolates, previously confirmed as positive on BioMerieux media as negative.

There was also a marked increase in false positives on Oxoid Brilliance™ agar in comparison to bioMerieux chromId™ upon incubation for a further 24hrs. An inoculum effect may have explained the differencesin the sensitivities obtained for both media tested, since one swab was used toinoculate both selective agars. However, this would haveaffected all results equally. Since swabs were inoculated in a manner to reduce such bias, Oxoid Brilliance™ MRSA lack of sensitivity during this trial was constant, with no increase or decrease in its sensitivity for MRSA detection based upon the sequence of inoculation. MRSA was isolated from 43 (12.7%) of the 336 specimens that were screened. BioMerieux chromID™ detected 43 (100%) MRSA isolates compared to only 27 (67.5%) MRSA isolates, that grew on Oxoid Brilliance™. (Table 4.1 Results)

f the 43 MRSA isolates recovered on bioMerieux chromID™ agar, >90% grew after overnight (20h) incubation.Oxoid Brilliance™ failed to recover MRSA from 13 MRSA confirmed specimens.

5.2 Disparity /Inconsistency

BioMerieux agar allows for the differentiation between MRSA and non-MRSA colonies that may both give rise to green colonies by the difference in colonial morphology and degree of the green colour. Oxoid's agar, on the other hand, provided no distinguishing factors that allowed for the differentiation between MRSA and non-MRSA isolates see figure 9-11 appendices.

Distinguishing between colonies that yielded blue / green colonies on each medium, but may not have been MRSA, was another factor in reducing the cost of further testing and decreasing the turnaround time (TAT) of result reporting/authorization.

Small pale green colonies, mainly after 48h incubation on bioMerieux agar were easily distinguishable from MRSA strains-large vivid green colonies, but no distinctive factors were evident with Oxoid's media, with non - MRSA colonies exhibiting comparable colonial morphology and colour to MRSA.

Another issue encountered with Oxoid's medium was that many colonies, though not producing the denim blue colour, were to be later confirmed as MRSA on bioMerieux chromID™ (at 20/48hr). Many, ultimately were positive for Oxoid Brilliance™ , but at >48hr, alerting to the fact that the media was not performing as desired and not as sensitive to MRSA detection as promised with a sensitivity of 67.5% compared to 100% for BioMerieux.(Table 4.5.2 Results

5.3 Ease of use

At GUH, swabs for inoculation were allocated one half of each plate.. It was found that since BioMerieux agar is a clear medium, it was easy to see where to inoculate, whereas, with Oxoid Brilliance™, being an opaque medium, it was not. This could lead to cross contamination between swabs inoculated and thus lead to an increase in false positives. This may seem trivial, but since the volume of specimens received daily was substantial, it would certainly be more cost effective not to allocate a single pate for each patient swab

5.4 Cost effectiveness

Although, theoretically, Oxoid's agar would be more cost effective - €0.80c versus €1.04 Biomerieux per plate - in reality this would not be the case, since, as observed in this trial, the tendency of the agar to allow growth of microorganisms other than MRSA (yielding blue colonies) leads to unnecessary time consuming and costly further confirmatory testing.

5.5 Findings

The yields of MRSA from various anatomical sites were dissimilar for the two media. From a total of 325 swabs and 11 ATCC strains, inoculated on both media, bioMerieux chromID™detected 43 MRSA isolates, Oxoid Brilliance™ detected only 27.

The number of specimens with suspicious colonies requiring further investigation which turned out not to be MRSA (false positives) differed substantially between the two media, with 9 of 336 (2.6%) for Oxoid Brilliance™ compared to only 3 of 336 (89%) for bioMerieux chromID™.

Senitivity was resulted as 67.5% and 100%, and Specificity was 96.9% and 98.9% for Oxoid Brilliance™ and bioMerieux chromID™ respectively.

Taking into account all of the above it seems that sensitivity and specificity are in all likelihood the most valuable of the indicators in the comparison between both agars for MRSA isolation. Biomerieux chromID™ agar was found to be easier to interpret, more specific and had better sensitivity than Oxoid Brilliance™ for MRSA detection.

6.0 Conclusion

Due to the importance of MRSA screening in a time of increased nosocomial and community acquired MRSA infections, the use of a media that is sensitive in its ability to detect MRSA infection, is vital in the rapid administration of treatment and thus, the decrease in its spread.

The tendency of Oxoid's Brilliance™ medium to facilitate growth of non MRSA isolates, with little or no differentiation to true MRSA isolates, and more significantly, the number of confirmed MRSA isolates it failed it identify during this trial, makes this medium unsatisfactory in respect to its use as an MRSA selective chromogenic agar.

For these reasons, although Oxoids Brilliance™ agar should, theoretically, detect MRSA earlier then the media currently in use, its lack of specificity and more so sensitivity were not advantageous as an alternative screening tool for the detection of MRSA.

In comparison, BioMerieux chromID medium was easy to interpret; had a high sensitivity even after 24h (>90%) and required less confirmatory testing, consequently reducing costs and TAT in result authorization.

Consequently, I would recommend the continued use of Biomerieux chromID™ MRSA agar, for culture based MRSA screening.

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