is the chromagar method more accurate for screening patients for methicillin-resistant...
DESCRIPTION
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.TRANSCRIPT
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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).