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Is the CHROMagar Method More Accurate for Screening Patients for Methicillin-Resistant Staphylococcus aureus Compared to Traditional Culture and Sensitivity?

Abstract

Purpose is to find out if CHROMagar method is more accurate for screening patients for

MRSA compared to traditional culture and sensitivity. Two studies were compared to find an

answer to the question. One study was done by Han ZL, E. Lautenbach, et al and the other by

Rahbar M, P. Islami, et al, where they compared CHROMagar MRSA to various media. Results

of both studies indicated the CHROMagar MRSA is equally the same for screening for MRSA

than traditional methods. The sensitivity and specificity of CHROMagar compared to these

traditional cultures are the same, where it is close to 100%. Traditional test such as the E-test and

Mannitol salt agar with Staphaurex slide test confirmation proved to be as accurate as the

CHROMagar MRSA in screening for MRSA. However these two studies varied greatly from

each other in terms of specimen size, the details of the procedure, and the statistics used to

support the results. The study written Han ZL, E. Lautenbach, et al. had all the factors stated

above, but the study written by Rahbar M, P. Islami, et al. lacked a large specimen size, had a

poorly written procedure and did have enough statistics to support it results, which made their

study unacceptable to support the research question.

Introduction

Of all the different species of the genus Staphylococcus, the one that is most dangerous if

not treated is Staphylococcus aureus (S. aureus). S. aureus can cause simple skin infection such

as pimple and boils to more serious wound and deep tissue infection. In wound and deep tissue

infection, the organism secretes toxins and certain enzymes that harm the host that may lead to

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shock or death if untreated. S. aureus is part of the normal flora in certain sites of the body but

the transfer of the organism due to trauma or person to person contact to sterile sites of the body

can cause an infection. Certain characteristics that help microbiologist identify S. aureus are: a

positive Gram staining that has cocci morphology, a unique yellow growth characteristic on

mannitol salt agar plates, and it being positive certain extracellular-enzymes like catalase,

coagulase, DNAase, lipase, and phosphatase .1

Treatment of S. aureus infection was penicillin, but it is not used anymore due to the fact

that most strains of S. aureus that evolve are resistant to penicillin. Penicillinase-stable

penicillins, such as oxacillin and methicillin, are used to treated infection caused by S. aureus

because the organism is still susceptible to these antibiotics. As new strain of S. aureus begins to

evolve some strains develop a mecA gene that produces a distorted penicillin-binding protein

making these strains of S. aureus resistant to penicillinase-stable penicillins. The strain of S.

aureus that is resistant to penicillinase-stable penicillins is called Methicillin Resistant

Staphylococcus aureus (MRSA). There are a limited number of drugs used to treat MRSA such

as vancomycin, linezolid, and daptomycin .2

Since MRSA is highly contagious, hospitals enforce preventable measures for workers

and patients to keep MRSA from spreading. Preventable measures includes, workers using

personal protective equipments such as gloves, gowns, mask, etc. It also includes the use of hand

sanitizers, washing of hands with soap and water, and proper cleaning of the hospital rooms and

equipment. Isolation of patients with MRSA is important to keep MRSA from spreading to

healthy people.3

Hospitals also screen patients that are positive for S. aureus for MRSA so they can treat

the infection using the right antibiotics and take the proper precaution if the patient is positive for

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MRSA. There are a couple of methods that the Clinical and Laboratory Standards Institute

(CLSI) recommends for the screening of MRSA. The three tests recommended for the screening

of MRSA are the oxacillin screen agar, cefoxitin disk screen test, and latex agglutination for

PBP2a .2

For oxacillin screen agar and cefoxitin disk screen, a .5 McFarland standard inoculum of

the specimen, which has previously been identified as S. aureus, is made to see if the strain is

resistant to penicillinase-stable penicillins. In oxacillin screen agar, specimen is inoculated in

Mueller-Hinton agar plates, which contains 4% sodium chloride and 6 µg/ml of oxacillin. The

inoculated oxacillin agar is left for 24 hours at 30-35ºC and if there is a growth after, then the S.

aureus is resistant to the oxacillin and the test is positive for MRSA.4

In the cefoxitin disk screen test, specimen is inoculated in Mueller-Hinton agar plates

containing a 30 microgram cefoxitin disk where it is incubated for 18 hours at 35ºC. If the zone

of inhibition is <21mm, then the strain of S. aureus is resistant to the cefoxitin and the test is

positive for MRSA. Cefoxitin disk screens are used instead of oxacillin because cefoxitin gives

clearer endpoints and is better at detecting the mecA gene in S. aureus.4

In the latex agglutination test, the antibodies present on the latex particle are against the

distorted penicillin-binding protein (PBP2a). This PBP2a is produced by MRSA making this

strain of S. aureus resistance to penicillinase-stable penicillins. The procedure of the latex

agglutination involves, sub-culturing the specimen in Colombia agar for 18 hour at 37ºC,

extraction of the PBP2a from the colonies by subjecting a loop of colonies to extracting reagents

and heat, centrifuged and then testing the final supernatant against the latex particle sensitized

with antibodies against PBP2a. If there is agglutination after three minutes then the test is

positive for MRSA.5 This test is also known as the Staphaurex slide test.

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CHROMagar-MRSA is one of the latest methods used in hospitals to screen patients for

MRSA. The agar in CHROMagar-MRSA contains sodium chloride, cefoxitin, inhibiting agents,

chromogen mix, and chromopeptones. When a sample is inoculated on the CHROMagar-MRSA,

other organisms, such as, yeasts, gram-negative organisms, and gram-positive cocci growth are

inhibited by the inhibiting agent. If methicillin susceptible S. aureus(MSSA) is present in the

sample, then it will not grow in the presents of sodium chloride and cefoxtin. However, if MRSA

is present, it will grow in the presence of cefoxitin and it colonies will be colored mauve because

the organism has consumed the chromopeptones for energy and interacted with the chromogen

mix resulting in it colored colony. Other organism that maybe in sample that is not MRSA and is

not affected by inhibiting factors may grow on the plate, but the color of their colonies if they

use chromopeptones will vary from blue to blue-green. If the organism does not use the

chromogenic material then the color of their colonies will be white or colorless.6

The purpose of writing this paper is to address the question of whether the CHROMagar

method is more accurate for screening patients for MRSA compared to traditional culture and

sensitivity. In order to answer the question, two studies were analyzed to find an answer to the

research question. The first article, Evaluation of a New CHROMagar Medium for Detection of

Methicillin-resistant Staphylococcus Aureus, was found in the Pakistan Journal of Biological

Science, where it was published in 2008 and written by Rahbar M, P. Islami, et al.. The article

was chosen based on the fact that it was recent as last year and as the title suggests, the

researchers evaluated CHROMagar ability to detect MRSA.7 The second article, Evaluation of

Mannitol Salt Agar, CHROMagar Staph aureus and CHROMagar MRSA for Detection of

Meticillin-Resistant Staphylococcus Aureus From Nasal Swab Specimens, was published in

Journal of Medical Microbiology, where it was published in 2007 and written by Han ZL, E.

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Lautenbach, et al. This article was also chosen, because it met the criteria of being recent and

addressing the research question, where it compared different methods to detect MRSA

including CHROMagar.8 This paper will analyze the two studies and its’ approach to addressing

and the answering to the research question.

Specimen Size & Collection

Specimen size, how and where it is collected are important factors in any study. It is

important for a study for have the right amount of specimens to draw a valid conclusion. For

example, if there are not enough specimens, the researchers would not have enough data to draw

a valid conclusion.9 Knowing how and where the specimen was collected are also important, if

the readers ever needed to replicate the researchers work, they would known exactly where and

how to collect the specimen. For example, if a study collected samples to be plated and only

writes that ‘the samples came from bathroom’, it would be hard to replicate the study’s data

because it is not known exactly where in the bathroom the sample came from or how it was

collected. That is why it is important for researchers to explain exactly how and where the

specimen was collected.10

In the study written by Han ZL, E. Lautenbach, et al, there were two phases where

specimens were collected from October 2004 to May 2005. At the Hospital of the University of

Pennsylvania, phase I of the study collected specimens from patients located in the medical

intensive care and transplant unit. In phase II, the specimens were collected from the medical and

surgical intensive care. The researchers chose these places because these are locations where a

patient has a higher probability of developing MRSA. In phase I of the study, when a patient

was admitted to the medical intensive care or transplant unit, a nasal swab was collected at

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admission and every week afterwards until it was positive. There were 316 nasal swab specimens

collected from the medical intensive care and transplant units. In phase II, researcher collected

nasal specimens from patients admitted to either medical intensive care or surgical intensive care

and every week afterwards until it was positive. It is noted that there is no differences in the

collection of the specimen in phase I or phase II, the only differences between the phases is the

nasal specimens in phase II were collected in surgical intensive care instead of the transplant

unit. 340 nasal swab specimens were collected in phase II from the medical and surgical

intensive care.8 The number of samples from the population was not small making the amount of

samples collected appropriate for the study, where the researchers have enough samples to draw

a valid conclusion. The specimen collection procedure from this study is well design where

researchers pick places where they know they have a greater chance of finding a specimen with

MRSA.

On the other hand, in study done by Rahbar M, P. Islami, et al, specimens were consisted

of 97 samples from Milad hospital of Tehran, where the exact date of collection or collection

procedure was not given. 97 samples are too small to make a valid conclusion if these samples

were just random samples, but the researchers seem to know the identity of the sample. The

authors wrote that the samples of S. aureus used was well defined and even stated that of the 97

samples, 58 were MRSA and 39 were MSSA. The authors do not state how the samples were

collected or how the samples were determined to be positive for S. aureus. The authors did say

that all the sample have a special typing pattern, but does not elaborate on the special typing

pattern or even where the sample came from.7 If some type of gene typing was done, the authors

did not state it. Knowing the identity of the specimen is good because the researchers can

compare the results from the experiment to what the right answers are. But not explaining how

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the samples were collected or how they were identified to begin with, it makes replicating

Rahbar M, P. Islami, et al experiment hard or even impossible.

Explanation of specimen collection is important because it affects how well the

experimental data can be reproduced.10 In this case, the study done by Han ZL, E. Lautenbach, et

al had the better specimen collection or least they explained their procedure better to the point,

where their data can be duplicated. The study done by Rahbar M, P. Islami, et al. seem to have a

good procedure by knowing the identity of each specimen in the experiment, but did not explain

where the specimen came from or how they came to know the identity of the specimen. Both are

equal in terms of addressing the research question and their approach to specimen collection, but

based on having a better explanation of where and how the specimens was collected, the study

written by Han ZL, E. Lautenbach, et al is more valid than the study written by Rahbar M, P.

Islami, et al..

Procedure

Just like explaining how and where a specimen is collected in a study, explanation of the

procedure is just as important. A vague explanation of a procedure will lead to inconsistent data

reproduction, if the experiment were to be repeated. A good study will have a good well detail

procedure, where if they needed to, any other researcher can repeat. It is necessary, for

researchers to explain everything the happen during a study, where leaving anything out, will

lead readers to confusion and make it even harder to reproduce the same results.10

Han ZL, E. Lautenbach, et al compared three tests against each other, to test its ability to

detect MRSA. Mannitol salt agar is being compared with the newer method of CHROMagar S.

aureus and CHROMagar MRSA. In phase 1, 316 nasal specimens were used in evaluating

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mannitol salt agar to CHROMagar S. aureus in the detection of MRSA. In phase 2, 340 nasal

specimens were use to evaluate CHROMagar S. aureus against CHROMagar MRSA All the

technical specification of the material and method were given, as the researchers followed

guidelines from CLSI. Colonies positive for S. aureus, would be mauve on CHROMagar S.

aureus and CHROMagar-MRSA and it woulbe be yellow on mannitol salt agar. Tests with no

growth was re-incubated for another 24 hr and those positive would be sub-culture in blood agar

plates and identified with catalase test, Staphaurex slide test, tube coagulase test and Vitex 2.

Follow-up testing on false positives were done and the researchers had tested 32 samples of

MRSA and coagulase-negative staphylococci to show that CHROMagar Staph aureus and

CHROMagar MRSA did not have a high incident of false positives.8 This study provides a well

detailed procedure, even follow up testing were done, making it possible to reproduce their

results. As stated already, the study was done in two phase where each phase used a different set

of specimens. The two phases were not done simultaneous, the first phase was done and then the

second phase, because the researchers did not have CHROMagar MRSA from the manufactures.

The study is not necessarily affected by this, but if a person is going to evaluate different media

for detection of MRSA, then at least the same specimen should be used on all media, acting as a

control. Meaning that the specimen used to evaluate CHROMagar MRSA is not the same

specimen used to evaluate mannitol salt agar, because of the use of two phases.

Rahbar M, P. Islami, et al.’ study from Pakistan Journal of Biological Science was just a

comparison of four methods of detecting MRSA. The E-test MIC, Oxacillin screen agar, manitol

salt agar plus oxacillin and CHROMagar were being compared against each other. Rose to

mauve colonies on CHROMagar were considered positive for MRSA and colonies on Oxacillin

screen agar and Mannitol salt agar were considered MRSA because the specimens was known to

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be S. aureus by the special typing technique. All the technical specifications of the material were

given, as the researchers did each method as recommend by the manufactured. Each method was

not done independently from each other. All the methods were done at the same time. That is

they were inoculated and incubated at the same time. It is mentioned the samples were made

from pure cultures and that complementary testing was use to initially to detect S. aureus, but the

authors does not go into detail about it.7 It is important that the reader get an explanation of this

special typing technique, what it is or how it works in detecting S. aureus. Leaving out

information or not explaining the procedure clearly would make it hard to replicate the

experiment due to the authors’ inconsistency.

The study Han ZL, E. Lautenbach, et al did have a well thought out procedure when

compared Rahbar M, P. Islami, et al’ study. The study states the exact procedure, which can be

followed and repeated. The study done by Han ZL, E. Lautenbach, et al has a procedure the will

provide answers to the research question. Unlike the study from Rahbar M, P. Islami, et al.

where, the researchers did not provide a good explanation of what the exact procedure they

followed and where their procedure in general seems to have holes in it, meaning that there are

some parts of the procedure missing or not included.

Results

Han ZL, E. Lautenbach, et al presented their results clearly. Of the 316 specimens

collected in phase one, 51 were detected as S. aureus and of that 51, 19 were MRSA.

CHROMagar Staph aureus showed it had a higher sensitivity at 24 hours and 48 hours and

mannitol salt agar had higher specificities at the previous indicated hours with and without

Staphaurex slide test confirmation. Of the 340 specimen collected in phase two, 39 was MRSA.

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CHROMagar Staph aureus with cefoxitin disk confirmation having a higher sensitivity than

CHROMagar MRSA at 24 and equal sensitivity at 48 h. The specificity of both media combined

with the Staphaurex slide test was the same at 100 %. False positive at 24 hours and 48 hours

were indicated for second phase of testing, which were mostly coagulase-negative staphylococci.

For the first phase of testing, false positive on mannitol salt agar was due to coagulase-negative

staphylococci and bacillus species.8 These results shows that the CHROMagar MRSA, or

CHROMagar S. aureus plus the cefoxitin disk diffusion test are very efficient in detecting

MRSA from nasal swab specimens, but they have to be confirmed by Staphaurex slide test. The

test shows that the three tests that are being compared with about the same in terms of specificity

and sensitivity.

Rahbar M, P. Islami, et al showed that the E-test and the CHROMagar was able to detect

all samples that were MRSA and those that are susceptible to methicillin. That would give the

CHROMagar and E-test sensitivity and specificity of 100%. Manitol salt agar plus oxacillin had

sensitivity and specificity of 95 and 100%, respectively, while oxacillin screening agar method

had 100% sensitivity and 95% specificity. Oxacillin screening agar had 2 false positive for

MRSA and Mannitol salt agar could not detect 2 stairs of MRSA. These result seems to correlate

with the results of the other study where, but these results cannot be trusted because of the many

holes that the procedure have. This study seems to have many ambiguities that make trusting the

results hard to do.7

These two studies showed that the CHROMagar method is not much better for screening

patients for MRSA as compared to traditional culture such as the E-test and Mannitol salt agar

with Staphaurex slide test confirmation. The sensitivity and specificity of CHROMagar

compared to these traditional cultures are the same, where it is close to 100%. Two different

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studies above proved that CHROMagar, E- test, and Mannitol salt agar with Staphaurex slide test

are all about the same in detecting MRSA. However, only the study done by Han ZL, E.

Lautenbach, et al can be used because it is more supported by facts and does not have any

discrepancies in it procedure. .

Statistics

Statistics are important part of a study, where researchers often depend on statistics to let

readers know the chances of random errors occurring during an experiment. It is important for

researchers to take into account random error and uncertainty because it can affect the find

conclusion of the study (9). In this case, the researchers of both studies needed to let readers

know that the media they are testing on can actually detected MRSA and it was not mere

coincidence that it happened during the experiment.

To quantify uncertainty in the study by Han ZL, E. Lautenbach, et al, a 95% confidence

intervals were used. For sensitivity and specificity of each test, a corresponding confidence

interval was given and a P-value was also calculated to detect random chance of variation. In

order to validate their results, the researchers use frozen strains that were previously positively

identified as being MRSA and coagulase-negative staphylococci, and compared CHROMagar

MRSA and CHROMagar S. aureus results to similar studies. The results was a 100% sensitivity

and specificity, which was consistent with another study by Diederen et al. (2005), which

showed a 100% sensitivity and specificity in detecting frozen strains of MRSA.8

Rahbar M, P. Islami, et al. did not state if there was a use of a statistical method to

diagnose accuracy, quantify uncertainty or test reproducibility. Other than stating the sensitivity

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and specificity of each method, no other statistics were given. In a study of this caliber, some

statistic should have been provided.

Discussion

Han ZL, E. Lautenbach, et al. final conclusion was that CHROMagar MRSA, or

CHROMagar S. aureus plus the cefoxitin disk diffusion test are very efficient in detecting

MRSA from nasal swab specimens. This conclusion is very well supported by evidenced, as

described above. The researchers think that CHROMagar MRSA, or CHROMagar S. aureus

could be screening test and be confirmed using the Staphaurex slide test, which can increased

sensitivity and specificity to almost 100%. This study still have its flaws where each specimen

that was confirm for MRSA using the Staphaurex slide test was not confirm directly for mecA

gene using molecular methods. However, cefoxitin disk test has a high sensitivity and specificity

for detecting MRSA just as much as molecular methods, the need to confirm directly for mecA

gene using molecular methods is not needed. Authors stated that traditional methods, in this case

mannitol salt agar, is equally efficient as CHROMagar MRSA but does not state the advantage of

using CHROMagar MRSA over mannitol salt agar. The authors should have given a reason on

why laboratory mangers should buy or clinical laboratory scientists should use CHROMagar

MRSA instead of mannitol salt agar.8

Even though a different method was use, the study written by Rahbar M, P. Islami, et al.

came to the same final conclusion as the other study, where CHROMagar makes a good

substitute for detection of MRSA. CHROMagar had a sensitivity and specificity of 100%, so it

would be very accurate. The E-test also has a sensitivity and specificity of 100%, but the

advantage of using the CHROMagar over the E-test would is not addressed in this study. Would

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CHROMagar in the laboratory save time, money, or material? As with all studies, limitation

include the use of very concentrated inoculums of MSSA, when they are present in low

concentration in clinical samples, and the presence of coagulase-negaive staphylococci, which

can affect the CHROMagar sensitivity and specificity in clinical samples. Due to these

limitations, the results from this study cannot be trusted.7

Conclusion

CHROMagar method is not much more accurate for screening patients for MRSA

compared to traditional culture and sensitivity. Han ZL, E. Lautenbach, et al, who had a well

thought out procedure supports the notion the CHROMagar MRSA is equally efficient as

traditional methods, but did not state a good reason on why it is useful to replace traditional

methods with CHROMagar MRSA. Since CHROMagar MRSA is as effective as older methods

then another study would have to be done on the economics of using CHROMagar MRSA

compared to traditional methods. If there is an economically valid reason to replace older

methods with CHROMagar MRSA then a study should help convince laboratory mangers of the

benefits.

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References

1. Forbes B, Daniel F. Sahm, and Alice S. Weissfeld. Bailey and Scott’s Diagnostic Microbiology. St. Louis, MO: Mosby; 2002.2. CDC. Laboratory Detection of: Oxacillin/Methicillin-resistant Staphylococcus aureus. Available from: http://www.cdc.gov/ncidod/dhqp/ar_lab_mrsa.html. Accessed 2009 April 5.3. CDC. Information About MRSA for Healthcare Personnel. Available from: http://www.cdc.gov/ncidod/dhqp/ar_mrsa_healthcareFS.html. Accessed 2009 April 5. 4. Pakistan Antimicrobial Resistance Network. MRSA. Available from: http://www.parn.org.pk/index_files/MRSA.html. Accessed 2009 April 5. 5. van Griethuysen A, M. Pouw, et al. Rapid Slide Latex Agglutination Test for Detection of Methicillin Resistance in Staphylococcus aureus. J Clin Microbiol 1999;39(9):2789-2792.6. Becton, Dickinson and Company. BBL™ CHROMagar™ MRSA. Available from: http://www.bd.com/ds/technicalCenter/clsi/clsi-chromagarmrsa.pdf. Accessed on 2009 April 1.7. Rahbar M, P. Islami, et al. Evaluation of a new CHROMagar medium for detection of methicillin-resistant Staphylococcus aureus. Pak. J. Biol. Sci 2008;11(3):496-8.8. Han ZL, E. Lautenbach, et al. Evaluation of mannitol salt agar, CHROMagar Staph aureus and CHROMagar MRSA for detection of meticillin-resistant Staphylococcus aureus from nasal swab specimens. J Med Microbiol 2007;53(1):43-46.9. Looney, Stephen W, editor. Biostatical Methods. 1st ed. New Jersey: Humana Press; 2001.10. Neill, Ushma. How to write a scientific masterpiece. J. Clin. Invest.117:3599–3602 (2007).