**FTS-CDC-PHPPO**

**January 25, 2005**

**11:30 a.m. CST**

Coordinator Good morning, and welcome to today's conference call. All
lines are going to be in a listen-only mode during today's presentation.
Today's conference call is being recorded. If you have any objections,
you may disconnect at this time. Now I'd like to turn the conference
over to your host for today, Ms. Denise Korzeniowski. Ma'am, you may
begin.

D. Korzeniowski Thank you. Good afternoon. This is Denise Korzeniowski,
training associate for the National Laboratory Training Network,
speaking to you from the State Lab Institute in Boston, Massachusetts.
Welcome to our teleconference, "What's New in the 2005 Standards for
Anti-Microbial Susceptibility Testing? New Recommendations from the
Clinical and Laboratory Standards Institute, formerly known as NCCLS."
This program has been made possible by an unrestricted educational grant
from Ortho-McNeil Pharmaceutical.

After the program, each participants needs to register and complete an
evaluation form documenting your participation that helps us to continue
to bring high-quality training programs in a variety of formats. To do
this, go to http://www.phppo.cdc.gov//phtnonline/. The password is 2005.
When you have completed the registration and evaluation form, you will
be able to print your CEU certificate. California and Florida CEUs are
also available. You have until February 26^th^ to complete this
progress. These instructions are on your original confirmation letter
and the general handout. They will also be e-mailed to each site
representative, again, no later than tomorrow. If time permits, the end
of the program will be opened up for questions. You are on a listen-only
line. We cannot hear you; you can only hear us. If you experience any
problems with the line during the teleconference, please press \*0. This
will signal the attendant that you are having a problem. If there are
technical difficulties, please stay on the line.

Again, welcome and thank you for joining us. We have over 1,000 sites
across the United States and Canada. Today's speaker is Janet Fick
Hindler, who speaks to us from the UCLA Medical Center in Los Angeles,
California. Janet Hindler is a Senior Specialist in Clinical
Microbiology for the Division of Laboratory Medicine at UCLA Medical
Center in Los Angeles, California. She is working as a consultant with
the Association for Public Health Laboratories to develop and conduct
training on antimicrobial susceptibility testing. It is my pleasure to
introduce to you and to welcome our speaker, Janet Hindler.

J. Hindler Thank you very much, Denise, and thank you, participants, for
tuning in to hear our program today, "What's New in the 2005 Standards
for Antimicrobial Susceptibility Testing?" I know some of you are
listening from locations recently plagued by nasty weather, including
the Boston office that's coordinating this program. I certainly
appreciate all the effort you put out to attend this program today. Now
hopefully those of you that are listening have had the opportunity to
print copies of the handout, so you're looking at the handout or viewing
the PowerPoint presentation that we sent to you or that you downloaded
from the Web site. If you do not have that information, we will tell you
at the end where you can find that. But nevertheless, all of the
material that we are covering today is included in your handout or can
be downloaded from the Web. There's some supplemental information that
you may have noticed that you received on the disc or that's posted on
our Web site, and I'll refer to that later on today to help you see
whether or not some of this information may be useful to you in your
particular setting.

So if you'll turn to slide two, you will see the objectives for today's
presentation and hopefully, by the end of this hour and 15 minutes or
so, you'll be able to outline the major changes found in the current
NCCLS tables, the M100-S15. You'll be able to discuss the current
recommendations for ESBL testing of Proteus mirabilis. You'll be able to
list specific drugs suggested for testing and reporting with
Acinetobacter, Stenotrophomonas maltophilia and Burkholderia cepacia.
You'll be able to describe our current recommendations for testing
polymyxin B or colistin by MIC methods; to discuss the new standard
reference method for MIC testing of Neisseria meningitidis.

If you go to the next slide, you'll see a continuation of the
objectives. These are to state the latest recommendations for detecting
oxacillin resistance and/or vancomycin resistance in the staphylococci
side; to explain the performance of the D zone test now for inducible
clindamycin resistance in Group B and other beta hemolytic streptococci.
Finally, to identify the use of ATCC reference strains for routine
quality control versus quality assessment purposes. We're going to cover
all of these topics in the next hour or so.

Now if you'll move on to the next slide, I just want to reiterate to all
of you the current documents that we should be using in our laboratories
for routine susceptibility testing. As you heard Denise mention and I'll
mention to you again, the NCCLS name is becoming obsolete and being
replaced by the Clinical and Laboratory Standards Institute or CLSI. The
reason for this is as follows: when the NCCLS was established back in
the 1970s, it was primarily a national organization. But during the past
decade, the NCCLS has reached out globally and it was felt that it was
essential to change the name to better reflect the global scope of this
organization, so we're now all known as the Clinical and Laboratory
Standards Institute. This organization will continue to use letters
NCCLS for some time, but they will ultimately be phased out and replaced
by CLSI. Nevertheless, the tables for susceptibility testing that we
should now be using were those just released in January, 2005, the
M100-S15 version. These contain the quality control ranges, the
breakpoints for various drugs, the suggestions for drugs to test and
report. These are to be used with the companion documents that tell us
how to do the reference disk diffusion and MIC methods. You can see the
current documents that were published in 2003 for this regard are the
M2-A8 for disk diffusion testing and the M7-A6. We will see a new
version of these documents in 2006, as the documents that tell us how to
do testing are updated every three years in contrast to the tables that
are updated annually.

Now if you'll move on to the next slide, I just want to remind you about
some of the reference terms I'll be using throughout this presentation.
When I mention M100, I'm talking about these new tables, the M100-S15
version. The M2 relates to the disk diffusion standard for performing
the disk diffusion test. M7 relates to the MIC method. CLSI is
synonymous with the NCCLS. Now I'm trying to become more and more
accustom to using the letters CLSI as opposed to NCCLS; but after 33
years, it's a little bit difficult. So undoubtedly, you're going to hear
me slip on more than one occasion and revert back to using the
terminology NCCLS. You'll also see that NCCLS remains on some of the
slides; but hopefully, by next year at this time, we will all be
referring to these documents by the CLSI designation.

Let's move on to the next slide and start looking at some of the changes
in this current document. If you'll go so slide seven, I've included a
photograph of an actual page out of the M100-S15 standard. This is in
the beginning part of the document that lists the updated information
that has been included in the new tables. This is something that all of
you should review to see which of those topics are pertinent to
practices in your laboratory, so you can introduce those into your
protocols accordingly. I might mention, in the materials that we sent
you or for those of you that are downloading material from the Web,
there is a checklist. Basically this checklist suggests actions you may
want to take related to the current recommendations. You can go down
this list and use it when you're modifying your procedures in your
laboratory, so hopefully this will be useful for you. You don't need
that checklist to listen to the presentation today, but this is
something that you might want to look at after the presentation and
incorporate it into your protocol for deciding how you're going to go
about making these changes, again, those that are appropriate to your
particular setting.

Now let's move on to the next slide and talk about some of the general
changes that have occurred this year. You can see some of the additions
include a clarification of the incubation temperature range for the
reference methods described in the CLSI documents. We have more
information on testing and reporting daptomycin and telithromycin, some
newer agents that are now being tested in some laboratories around the
country. I'll talk a little bit more about this later. Finally, testing
the ATCC strains for quality assessment versus quality control purposes.

Now if you'll go to the next slide, let's look a little bit about this
issue of incubation temperature range. Previously a lot of you can
probably remember that we specified 35 degrees Centigrade as the
temperature for the various tests that we describe in our CLSI
documents. Now we've clarified this. As you can see on this slide, for
staphylococci, the specific range that we document here is 33 to 35
degrees Centigrade with a caveat saying that we should not exceed 35
degrees Centigrade. Now why is it different for staphylococcus than
other organisms? The reason for this is because, if we were to incubate
at temperatures higher than 35 degrees Centigrade, we may compromise our
abilities in being able to detect oxacillin or methicillan resistance
staph aureus and coagulase-negative staphylococci. So that's the reason
that we recommend not to exceed 35 degrees Centigrade when incubating a
reference test for staphylococci in our routine protocol. For all other
organism antimicrobial combinations, the incubation range is 35, plus or
minus 2 degrees, Centigrade. There are some rare exceptions when you
talk about some of the unusual organisms, such as the Campylobacter and
so forth, but I'm not going to talk about those today.

Now let's move on to the next slide and talk a little bit about
daptomycin followed by a little bit about telithromycin, because it's
important for us to know about these new drugs that have just been added
for the first time to our listing of drugs suggested for
testing/reporting in clinical laboratories. Now many of you know that
daptomycin is a lipopeptide agent. It can only be administered by the IV
route. It does have FDA approval for treating patients with complicated
skin and skin structure infections due to staph aureus, the beta
streptococci Group A, B, C and G, and vancomycin-susceptible
Enterococcus faecalis. If you were to look at the FDA package insert for
how this drug may be used, you would see these recommendations right
here in the package as listed on this slide.

Now let's go to slide 11. In terms of daptomycin testing, there are some
special testing considerations. This basically relates to the amount of
calcium in the medium necessary for this drug to perform optimally. You
can see, when we're talking about broth-dilution testing of daptomycin,
we have to make sure the medium contains 50 micrograms of calcium.
Otherwise, you may get erroneous results. Now currently disks for
testing are not yet commercially available, although it is my
understanding the Oxoid disk will become available next month. However,
you can procure these disks from the pharmaceutical manufacturer Cubist,
and I have included their telephone number on this slide 11.

In terms of MIC testing, MIC tests are not available on Trek panels,
PASCO panels and, also, with the Etest. The Etest recently has been FDA
cleared for testing daptomycin. Alternatively, if you had to have an
organism tested by MIC, you can submit it to Laboratory Specialist
Reference Lab, and they will do the testing for you. They will provide
you with the shipping materials and so forth. So if you're in a position
where that would be the best option for you, you can contact Laboratory
Specialist. Their phone number is listed right here.

Now let's look at the next slide where we've shown a little bit more
about the daptomycin testing issues. Here is listed the breakpoints for
disk diffusion and MIC testing for staphylococcus and enterococci.
What's striking here is that we only have susceptible breakpoints. The
reason for this is because, at the time these breakpoints were
established and today also, there are very few isolates of staphylococci
or enterococci that are other than susceptible to daptomycin. Daptomycin
is a very broad spectrum gram-positive agent and there are very few
organisms that are resistant to this particular agent.

Now you'll see in the little comment I have on this slide right here
that recently there has been some concern about the disk diffusion
method failing to identify isolates that have elevated MICs to
daptomycin. There have been rare occasions where there have been
staphylococci or enterococci that have MICs above the susceptible
breakpoint. Some of these have tested susceptible by the disk diffusion
methodology. This is something that is being investigated by CDC and
other researchers. At this point in time, we're not sure if there has to
be a disk change or if this is due to some components, actually the
content of calcium in the medium. But nevertheless, it's important for
everybody to be aware that we may see false susceptible results if we're
using the disk diffusion test in light of the fact that the disk
diffusion method may not be able to identify those isolates that have
decreased susceptibility to daptomycin. Probably we will be hearing more
about that in the near future. Optimally, the ideal method for testing
daptomycin would be by an MIC methodology at this time until this
problem is resolved.

Now let's move on to the next slide. This is an excerpt from our table
in our M100 standards. I think a lot of you are familiar with this
table, because it's existing in the M100 tables for the past couple
years. Basically this suggests those drug-bug combinations that we
should verify or those results that we should verify prior to reporting
out patient results. We've added daptomycin to this list and basically
we're stating that, for the enterococci, for staphylococci and for
streptococci, if we were to encounter any isolate that was other than
susceptible to daptomycin, we should pursue it. As I mentioned, it'd be
very, very rare to identify a strain of these organisms that was other
than susceptible to daptomycin. So here what you would do is confirm the
identification, repeat the susceptibility test to indeed make sure that
you are dealing with an isolate that is not susceptible to daptomycin.

Now if you'll go on to the next slide, I've just highlighted here what
are we going to be about testing and reporting daptomycin. It's my
understanding, at this point, because this is a very broad-spectrum drug
that's reserved for treating very serious infections where
narrow-spectrum agents would not be appropriate, it is not being tested
routinely in most laboratories; but, of course, this may change as more
and more become familiar with this agent and the need arises.
Nevertheless, each laboratory has to obtain input from the medical staff
when determining if and when you may want to test daptomycin in your
setting. This is not a decision that can be made in a laboratory alone,
because we have to make certain that we're providing information that
the staff will welcome.

Now in our CLSI M100 tables, in Table 1, it lists those organisms for
which it might be appropriate to test daptomycin, including
staphylococcus, Enterococcus; and there is a caveat for enterococcus
stating, for vancomycin-susceptible Enterococcus faecalis only. Then for
the streptococci, we also have a qualifier saying, "For beta hemolytic
streptococcus only." Now I know some of you may be asked to test
daptomycin on organisms other than those addressed in our NCCLS or CLSI
tables. Providing you get in writing from your medical staff that this
is something that they want you to do, I feel it would be appropriate
for you to test organisms other than those listed in our NCCLS
standards. But again, I personally feel that, if you're going to deviate
from the recommendations in our CLSI or NCCLS standard, it would behoove
you to get this in writing from your medical staff, so at least you have
documentation that this is something you've been asked to do above and
beyond what we're recommending in our current CLSI standards.

Now let's move on to telithromycin and talk a little bit about this new
agent that appears for the first time in our tables of suggested drugs
for testing and reporting against various organism groups. Telithromycin
is a ketolide agent. It's only administered by the oral route. It has
FDA approval for treating infections, bronchitis, sinusitis,
community-acquired pneumonia due to the pneumococcus, Haemophilus
influenzae, Moraxella cataralis, also for treating sinusitis due to
staph aureus, the pneumonias cased by the atypical pathogens, mycoplasma
and chlamydia and, also, multi-drug resistant streptococcus pneumonia.
Here, too, you can see that this agent is primarily used for treating
respiratory types of infections.

Now let's move on to the next slide. Again, we have to decide whether or
not we're going to be testing this agent in our laboratory routinely and
we have to get help from our medical staff to make this decision. Quite
often, this agent is prescribed empirically where it's really not
essential for laboratories to do routine testing. In the CLSI M100
tables, telithromycin is listed as drugs that might be considered for
testing against staphylococcus, Haemophilus; but I might mention with
Haemophilus, it has been included with that footnote where we state that
telithromycin maybe used for empiric therapy, but the test results are
often not useful for management of individual patients. Again, too, this
reflects the fact that telithromycin is often prescribed empirically for
respiratory infection. Also, telithromycin is listed under the
streptococcus pneumonia category as an agent that might want to be
tested and reported on this particular organism. So again, too, with
both of these newer drugs, each laboratory has to interact with the
medical staff to find out when it might be appropriate to test and
report these drugs on organisms isolated from patients in your
institution.

Let's move on to slide 17 and talk a little bit about this whole concept
of quality assessment versus quality control. I might mention that a lot
of us are familiar with the term "QA," which used to stand for "quality
assurance," but now CLIA has decided that QA actually stands for
"quality assessment." Nevertheless, here we want to talk about those
ATCC strains that we have now introduced into our M100 documents for
quality assessment in contrast to quality control purposes. So let's
talk a little bit about what this actually means. We talk about using
these ATCC strains for quality assessment. Basically we're talking about
using these for training purposes to help demonstrate the techniques and
learn how to do the test to ensure accurate results; for competency, to
make sure that each operator, each technologist performing the test,
does the testing correctly; and for test evaluation, if we're bringing a
new test on board, to determine the accuracy of results during the test
implementation, if we were to make any modifications in our procedure
and so forth. So as you can see here, we're using these strains that are
labeled as quality assessment strains to make sure that the test that
we're introducing actually works correctly and is performed correctly.

Now as an example of this, and we're going to talk a little bit more
about this later on, we can look at the D zone test. We have now
included two strains for quality assessment purposes, a negative-control
strain and a positive-control strain. I've listed their ATCC numbers on
the slide right here. So what we're saying is that you don't need to
test these weekly, like we do our standard quality control strains, but
we would want to include these or similar strains to make sure that we
know how to adequately perform testing.

Let's move on to the next slide and talk a little bit about the contrast
being quality control strains and using ATCC strains for quality control
purposes. I think we're all familiar with the fact that we use these
strains and test them either daily or weekly to ensure our test
methodology is producing accurate results. Acceptable results will imply
that the following are satisfactory; that our equipment is working
appropriate; that the reagents, the media and supplies are adequate, and
the person performing the test is doing so correctly. An example with
the D zone test of our quality control strains, we quality control the
erythro and clindamycin disks with our staph aureus ATCC 25923. Again,
I'm going to talk a little bit more about this in detail later. So I
hope you can understand this concept of these strains for quality
assessment versus quality control purposes.

Now let's move on to slide 19 and talk about the changes that have
occurred among the gram-negative bacteria in our M100-S15 document.
We'll start by looking at slide 20, where we talk about the changes for
enterobacteriaceae. Basically the primary changes that have occurred
involve ESBL testing of Proteus mirabilis and clarification of ESBL
testing quality control or quality assurance recommendations.

We go on to the next slide. I've summarized some of the issues related
to ESBL testing of Proteus mirabilis. Now a lot of us know that ESBLs
have been found in multiple species other than E. coli and Klebsiella.
Currently, at this time, the only additional organism that the CLSI is
recognizing is Proteus mirabilis. I might mention, however, that a lot
more work is being done right now at this whole issue of ESBL testing
and our ability to identify beta-lactam resistance among the
enterobacteriaceae. Nevertheless, for Proteus mirabilis, we're not
saying that you need to perform ESBL screening on this organism. We are
suggesting to consider the ESBL test when the organism is clinically
relevant, for example, if you had an isolate causing bacteremia. Now
here, too, like with all the other decisions in our laboratory, it's
important to interact with our medical staff to determine when it would
be appropriate for testing for ESBL and Proteus mirabilis in our
institution. One practical approach, at this time, would be to test
sterile body site isolates or we might want to consider also testing
those isolates that may look suspicious for producing an ESBL such that
these isolates have decreases susceptibility to some of the
extended-spectrum beta-lactam agents. This would suggest it might be
suspicious for an ESBL producer.

Now we use the standard ESBL screen test with a slight modification.
Those modifications are listed on slide 22. So you can see the indicator
drugs that we've used for years for the E. coli and the Klebsiella. Now
you can see which ones are applicable for screening Proteus mirabilis,
those being cefpodoxime, ceftazidime and cefotaxime. As far as the disk
screen is concerned, there's really no difference in the zone
breakpoints that are used for screening for ESBL production in Proteus
mirabilis as compared to the E. coli and the Klebsiella. When we looked
at the MIC screen, the only change that has occurred for Proteus
mirabilis is the breakpoint for cefpodoxime. Here for Proteus mirabilis,
we're going to use a breakpoint of greater or equal to two in contrast
to using greater or equal to eight for E. coli and Klebsiella. As you
can see here, too, at this point in time, we're not recommending the
aztreonam or ceftriaxone be used for ESBL screening.

Now if we go to the next slide, what about confirmation testing or the
phenotypic confirmatory test for ESBLs? Basically we use the same disk
diffusion or MIC ESBL phenotypic confirmatory test without any
modification. So we can perform the confirmatory test for Proteus
mirabilis in identical fashion to the way we're performing this
phenotypic confirmatory test of E. coli and Klebsiella.

Now let's move on to the next slide. One of the reasons why we're saying
it's not essential to screen all Proteus mirabilis is in light of the
fact that this occurrence is very uncommon in the United States. There
are several papers that I have referenced in your handout and I've just
cited one of them on the slide right here. This is data that was
generated from a group at CDC, Dr. Schwaber and colleagues. Basically,
in that study, they showed that looking at 171 Proteus mirabilis
strains, only 1 of those was confirmed to be ESDL positive with a TEM
type ESBL enzyme. However, the incidence of ESBL producing Proteus
mirabilis is substantially higher in some parts of the world. There have
been significant problems with ESBL-producing Proteus mirabilis in some
European countries and South America and so forth. At this point in
time, it's not a severe problem in the United States, but it's really
important for clinical laboratories to prepare to deal with this and
identify ESBL-producing Proteus mirabilis, particularly in isolates
causing serious infections, such as bacteremia.

Now let's move on to the next slide. The other issue that we've expanded
upon in light of the enterobacteriaceae also deals with the ESBL test.
Now we've clarified when it's essential to do quality control versus
quality assessment for both the screen and the phenotypic confirmatory
test. For the screen test, we've now described that, for daily or weekly
quality control, we can test the ESBL-producing Klebsiella, the ATCC
700603 or the E. coli ATCC 25922, which is our standard E. coli quality
control strain. So for the screen test, it's only necessary to test one
of these daily---most of us are doing weekly quality control, so this
would really mean that most of us will be testing either of these
strains weekly with the screen disks.

For quality assessment purposes, we can use the ESBL-producing strain. A
practical way would be to test your E. coli on a weekly basis. However,
make sure the screen works periodically and your staff is competent in
doing the screen by testing the ESBL-producing Klebsiella 700603 strain.
In contrast, for the phenotypic confirmatory test, we're suggesting that
both the ESBL-producing Klebsiella and the E. coli be tested daily or
weekly and included in part of your quality control protocol.

I might mention there's a little footnote on this slide. This relates to
the instability of the plasmid on which the ESBL gene is located. It's
been shown in several occasions that, if these strains were stored at
temperatures above minus-60 degrees, the organisms can lose the plasmid
and lose the gene and, therefore, your ESBL-producing Klebsiella would
revert to a susceptible strain, and you might have problems with your
quality control. So you have to be careful about storage of this strain,
if you're indeed storing these strains in the freezer.

Now let's move on to the next slide and talk a little bit about some of
the changes with the non-enterobacteriaceae. The primary addition here
is we've clarified and separated out the drugs recommended to test and
report for Acinetobacter, Burkholderia cepacia and Stenotrophomonas
maltophilia. We've also added some recommendations for MIC testing for
polymyxin B and colistin. We've actually added disk diffusion
interpretive criteria for Trimeth Sulfa and Burkholderia cepacia.

Now if you go to the next slide, I've included a photograph of the tops
of the table one from both the disk diffusion and the MIC documents. The
thing I'd like to highlight here is the second column, and you can see
the arrow pointed to that second column. For the disk diffusion test, we
can see that the column header specifies Pseudomonas aeruginosa only. In
contrast, the column header for the MIC test specifies Pseudomonas
aeruginosa and other non-enterobacteriaceae. This reflects additional
non-enterobacteriaceae can be reliably tested by the MIC method, but not
the disk diffusion method.

If we go to the next slide, this is just a photograph of the expanded
table for drugs recommended for testing and reporting on the
Acinetobacter and Burkholderia and Stenotrophomonas. This is actually
out of the disk diffusion document. There is a very similar table with a
few additional drugs on the MIC document as well. But nevertheless, the
point here is to make sure that you're aware that we now have clarified
those drugs recommended to test and report against the Acinetobacter,
Burkholderia and Stenotrophomonas maltophilia.

Now if you go to the next slide, I just want to clarify---and again,
too, I've reposted the header for the column in the MIC, or M7 document,
stating that here the header includes Pseudomonas aeruginosa and other
non-enterobacteriaceae. There's a footnote stating that the other
non-enterobacteriaceae include Pseudomonas species, such as ..., and
nonfastidious, glucose-nonfermenting gram-negative bacilli with the
exception of the Acinetobacter, Burkholderia and Stenotrophomonas,
because we now have separate lists for these three groups of organisms.
Here, too, it's important to understand which particular organisms are
included in that non-enterobacteriaceae category.

If you go to the next slide, there is another footnote that relates to
drugs that may be appropriate in testing on other non-enterobacteriaceae
other than Pseudomonas aeruginosa. The actual footnotes reads that these
drugs may be indicated for routine testing of some Pseudomonas species
and other nonfastidious, glucose-nonfermenting gram-negatives with the
exception of Pseudomonas aeruginosa. Here we've listed Ticar/Clav, Trim
Sulfa, cefotaxime or ceftriaxone, Chloro and then Ceftizoxime and Tetra
when reporting on isolates from urine cultures. Now the important point
here to remember is that what we're saying in Table 1 are suggestions
for routine testing and reporting. It doesn't necessarily mean these
drugs should never be tested on these other organisms, but basically
we're referring to which drugs you might consider for routine testing
and reporting protocols in your laboratory. So even though you may see,
for example, interpretive criteria for Ticarcillin-clavulanic acid in
Pseudomonas aeruginosa, what we're specifying in Table 1 is that most
laboratories would probably not consider this as a primary or a routine
agent. They'd want to report on Pseudomonas aeruginosa. So it's
important to understand that distinction between how we list drugs in
Table 1 and 1-A as compared to Table 2 that contains the breakpoints.

Okay, let's move on to slide 31. On this particular slide, I've
indicated a susceptibility profile for Acinetobacter baumannii. Probably
some of you have seen this problematic organism in your laboratory that
basically is resistant to all of the drugs on our panel. This pretty
much represents the drugs that are recommended for treated Acinetobacter
infections.

So if you go to the next slide, what we need to think about when we see
an organism that's resistant to all of the drugs on our panel, first of
all, we need to consult with the physician ordering the test to make
sure he or she has consulted with an infectious disease specialist, of
course, if it wasn't an infectious disease specialist who actually
ordered the test in the first place and is familiar with it. Then the
other questions we have to consider: is this isolate important to
patient management? In a laboratory, in most cases, we cannot determine
this. That's why it's so essential for the physician that's treating the
patient get some help in evaluating whether or not this is an organism
that might be colonizing the patient and wouldn't need therapy as
compared to an isolate that's probably infecting the patient where
therapy would be necessary. The other thing, too, is to remember that
we're not only concerned about possibly finding an agent that might be
appropriate for treating this patient; but also, should this organism
spread to other individuals in our healthcare facility, we'd want to
have an idea of what drugs might be appropriate.

Now how are we going to test some of these supplemental agents, because
not all of us keep every possible drug in our laboratory for testing?
This might represent an opportunity to interact with the reference lab
that can test some of the supplemental agents that might be appropriate.
Also, more recently, we've added recommendations for testing the
polymyxin or colistin. Among these highly resistant Acinetobacters and
also the highly resistant Pseudomonases, these are the only agents that
are appearing active against these strains.

I've included on this slide right here a resource that might be helpful
to those of you that are working on cultures from patients with cystic
fibrosis. This is the Cystic Fibrosis Referral Center for Susceptibility
and Synergy Testing at Columbia in Manhattan. This laboratory is
directed by Dr. Lisa Saiman. They do offer synergy testing and
sophisticated testing of highly resistant isolates from patients with
cystic fibrosis. We're not saying to send every isolate from a cystic
fibrosis patient to this particular reference laboratory, but it's
important to know this resource exists, because there may be occasions
when it may be helpful to have supplemental testing done. Dr. Saiman's
lab is likely going to be able to help you.

Let's move on to the next slide and talk a little bit about the addition
of polymyxin B and colistin to our MIC testing document. Now we have
listed in M100-S15 both MIC and disk diffusion quality control ranges.
However, there are only MIC breakpoints for polymyxin B. There is a
comment stating that polymyxin B MIC results can predict colistin MIC.
Basically the breakpoints for polymyxin are two or less are susceptible;
four or greater is resistant. If you were to have an isolate susceptible
to polymyxin, we could conclude that it would be susceptible to
colistin. If it were resistant to polymyxin, it could be considered
resistant to colistin. So here, too, we would test polymyxin and then
include a comment about how this would relate to the activity of
colistin, because colistin is more likely the drug that would be
prescribed to the patient among this class of agents.

Now a lot of you out there are probably wondering how would you be able
to do this testing, and we do not have disk diffusion breakpoints at
this time. There is a reevaluation of the disk diffusion test being
investigated. However, it's unlikely that the disk diffusion test is
going to be satisfactory to testing any of the organisms that we may
wish to test. But again, at this point in time, this hasn't been
completely ruled out. There are problems with disk diffusion testing and
the ability of the disk diffusion test to detect resistance. So at this
point in time, the best methodology for testing polymyxin would be to
use a MIC method. Unfortunately, at this point in time, to the best of
my knowledge, there is not a commercial method for doing MIC testing of
polymyxin, but we know commercial manufacturers are looking into this at
this time. So if you had to have an isolate tested for polymyxin, the
ideal solution would be to submit it to a reference lab that can do a
viable MIC method for you.

I might mention, too, in both Acinetobacter Pseudomonas, resistance is
relatively uncommon at this time, but there is concern about increasing
resistance. So probably in the near future, many of us are going to be
doing testing of polymyxin on these highly resistant organisms.
Interestingly, there were breakpoints for polymyxin and colistin back in
the 1970s. However, when less toxic broad-spectrum agents were
introduced in the 1980s, it was felt that colistin and polymyxin were no
longer needed. But now that we've seen resistance to emerge to these
broader-spectrum agents that were introduced in the 1980s, such as your
third-generation ..., the colistin polymyxin group remains an only
alternative in treating infections caused by some of these highly
resistant organisms. So you're probably going to see more and more about
colistin and polymyxin in the near future.

Let's move on to the next slide and look at another important addition
to our M100 table and that is a standard reference MIC method for
testing Neisseria meningitidis. This is something that all of us had
welcomed for a long time. Thanks to our colleagues, Dr. Jim Jorgensen
and his laboratory down at the University of San Antonio, they have
developed a standard MIC reference method for testing Neisseria
meningitidis. On slide 34, I've just included a photograph of that table
in our CLSI document.

If you go on to slide 35, I've just recapped what this methodology
involves. Basically, for inoculum preparation, we're going to use the
direct colony suspension method, the same method that we'd use for other
fastidious organisms. It's recommended to select the growth from
chocolate agar that has been incubated for 20 to 24 hours. Incubation is
at 35 degrees in an atmosphere of increased CO~2~ for 20 to 24 hours.
The broth medium actually used is the same broth medium we recommend for
testing pneumococcus and the streptococcus species; that being the
cation-adjusted Mueller-Hinton broth with 2% to 5% lysed horse blood.
For quality control, the recommendations are to use the standard
pneumococcus, the 49619, and also the E. coli for select agents.

Now there is a little comment on this slide right here indicating that
you need to take very serious safety precautions when testing this
organism in your laboratory, ideally doing the testing in your biosafety
cabinet, because, as you know, when you are making inoculus extension
and so forth, you may create aerosols. I think a lot of you out there
are familiar with some of the problems that have been experienced with
laboratory-acquired meningococcal infections. We certainly want to avoid
those in our particular laboratories, so be very careful if you're
working with this organism.

Now if you go on to the next slide, I've just listed those drugs that
are included in the table as therapeutic agents that might be used for
treating meningococcal disease, those being penicillin, ampicillin,
cefotaxime, ceftriaxone, meropenem and chloramphenicol.

If you go to the next slide, in contrast, there are some drugs with
interpretive criteria or breakpoints for agents that would be used in
prophylaxing individuals that have come in contact with a patient with
meningococcal disease. These agents are not used in treating
meningococcal infection; but again, used for prophylactic purposes;
those being azithromycin, cipro, levo, minocycline, rifampin,
sulfisoxazole and trimethoprim-sulfamethoxazole.

Now if you go to the next slide, just a very brief summary of some of
the resistance concerns in Neisseria meningitidis. Basically the
resistance that has been reported to date includes rare beta
lactamase-producing isolates. Now the first of these were identified in
the early 1980s and there was great concern about beta lactamase
production being a common occurrence of meningococci, but fortunately
that hasn't been the case. To date, there have been only six cases of
beta lactamase-producing meningococci identified in literature. The most
recent was nearly a decade ago and that was in Spain. So we're not
saying you need to do beta lactamase testing routinely on the
meningococci, but it is important to be aware that these observations
have existed in the past.

Now there are some strains that will be categorized as penicillin
intermediate or resistance where the mechanism here is altered
penicillin-binding proteins. However, these isolates remain very
susceptible to the extended-spectrum cephaolosporins that are often used
in treating meningococcal disease. Also, it seems, too, that if a
patient were infected with one of these strains, by prescribing the
standard high doses of penicillin recommended for treating meningococcal
disease that these patients would respond. This is something, again, to
be concerned about for practical purposes. It has not seemed to impact
significantly on the recommended therapy for treating meningococcal
disease.

In terms of resistance to the prophylactic agents, it's very common to
encounter resistance to the sulfonamides. There have been rare
resistance reported to cipro, and resistance to rifampin is very
uncommon. There may be occasions when you may be asked in the laboratory
to test meningococci against the agents for prophylactic purposes as
well. Now in terms of the strategy for dealing with the testing, we're
not advocating that you test every meningococcus you encounter in the
laboratory. This is probably something that should just be tested upon
physician request.

If you move on to the next slide, we'll start talking a little bit about
some of the changes related to testing the gram-positive bacteria. On
slide 40, we've just summarized what those are. Basically we've expanded
some recommendations for dealing with oxacillin testing reporting by
either looking at the mecA or PBP2a testing, using the cefoxitin disk
diffusion test, reporting beta lactam results of oxacillin-susceptible
staphylococci and then testing Staphylococcus lugdunensis. We've also
included some expanded recommendations for identifying staph aureus with
reduced vancomycin susceptibility by using the BHI vanco screen agar.

If you go to the next slide, a continuation; we've clarified the
recommendations for quality assessment and quality control from the
clindamycin induction or D zone test. We've revised some disk diffusion
and MIC breakpoints for the fluoroquinolones and staphylococci. I've
already talked about some of the additions that we've made reflecting
daptomycin being added to our NCCLS tables or our CLSI tables. Then
we've also added telithromycin as a potential drug that might be tested
in select circumstances against the staphylococci.

Now let's move on to slide 42 and recap the oxacillin-resistant concerns
among the staphylococci. I think all of you are probably aware of the
mecA gene being responsible for oxacillin resistance is what we perceive
as the classic type of MRSA and methicillan or oxacillin resistance
coagulase-negative staphylococci. We know that the mecA gene code for
penicillin-binding protein 2a, which confers the oxacillin resistance.
We know that there are tests out there for mecA or the PBP2a, and these
will detect that classic type of oxacillin resistance. The majority of
oxacillin-resistance staphylococci that we encounter are resistant due
to the presence of mecA and PBP2a.

Now in the past and even today, there are very rare reports of
oxacillin-resistant staphylococci that are mecA or PBP2a negative. This
is primarily in staph aureus. These have been referred to as borderline
oxacillin-resistant strains and these occasionally have MICs or zones in
the resistant range. Now we don't have time to go over this in great
detail today. I just included these on the slide, because I know some of
you are encountering some of these strains in your laboratory and often
there is a dilemma in terms of how to further test and report these
agents. Hopefully in the near future, we'll be able to address that more
completely.

Now let's move on to the next slide and discuss a little bit the
phenotypic tests for mecA-mediated resistance in staphylococci. Here
we're talking about those tests that can detect the classic type of
oxacillin or methicillan resistance. In terms of disk diffusion testing,
we're now recommending using the cefoxitin disk as a surrogate for
predicting oxacillin susceptibility or resistance. Now as far as staph
aureus is concerned, the results of the cefoxitin disk diffusion test
are comparable to the oxacillin disk diffusion test. For
coagulase-negative staphylococci, the results of the cefoxitin disk are
better than the oxacillin disk.

The other thing, when reading results for the cefoxitin disk, we
recommend using reflective light. This is contrast to what we previously
recommended for reading the oxacillin disk test, where we suggested
looking at that zone very closely using transmitted light. It is much
easier to identify oxacillin resistance by use of the cefoxitin disk
than by use of the oxacillin disk. It's important to remember, however,
we're just using the cefoxitin disk as a surrogate for detecting
oxacillin resistance. So we do not report out cefoxitin; we report out
oxacillin. As far as MIC testing is concerned, at this time, it has not
been demonstrated that using cefoxitin is superior to using oxacillin,
so we still use oxacillin in our MIC test system.

Let's move on to the next slide and talk about the interpretive
criteria, or breakpoints, for the cefoxitin disk for mecA-mediated
resistance in the staphylococci. There has been a change here.
Previously staphylococcus lugdunensis was grouped with the
coagulase-negative staphylococci, but now it has moved up to the
category that includes the staph aureus. So basically we're using the
same cefoxitin zone breakpoints for lugdunensis as we used for staph
aureus. There has not been a change for coagulase-negative staphylococci
and the actual zone diameters that would identify cefoxitin resistance
or susceptible is the same. Here you can see, too, again, we're
reiterating that, if you saw cefoxitin resistance, you would report the
organism as oxacillin resistant. If you saw cefoxitin susceptibility,
you would report the organism as oxacillin susceptible.

Now let's move on to the next slide. Some have asked "Why does the
cefoxitin disk work better than the oxacillin disk in detecting isolates
with mecA?" It's been shown by various high-level investigators that the
mecA expressed a higher level in the presence of cefoxitin as compared
to oxacillin. So that's why we have decided that this is a better
mechanism for identifying oxacillin resistance than using the oxacillin
disk itself.

Now let's move on to the next slide and talk a little bit about the
abilities of these various tests that we've described to be able to
detect mecA-mediated resistance in the staphylococci. On slide 46, we're
going to talk about these in terms of sensitivity and specificity. On
this slide, I just wanted to review with you what we mean by the
sensitivity of a susceptibility test and its ability to detect
susceptibility or resistance. We're going to talk about the use of these
various tests. We're going to talk about sensitivity as the ability of
these tests to detect resistance. To calculate the sensitivity, we
basically look at the number of resistant results divided by the number
of mecA-positive strains times 100. Here we're using mecA as the gold
standard for identifying oxacillin resistance. For specificity, what
we're saying is that we're looking at how well this test is not going to
report false resistance. To calculate this, we look at the number of
susceptible results divided by the number of mecA-negative strains times
100.

Now let's look at the next two slides. I'm very appreciative of Jana
Swenson, Fred Tenover and Jean Patel for providing these data to us. If
we look at the sensitivity of the various test methodologies for staph
aureus, basically the oxacillin MIC and the oxacillin disk diffusion,
the cefoxitin disk diffusion and the PBP2a all performed comparably to
our gold standard, which is mecA. The sensitivity is very high, so all
of these methods do very well in detecting oxacillin resistance.
Similarly, in terms of specificity and the ability of these methods not
to call an organism falsely resistant is very, very good. The oxacillin
MIC, oxacillin disk diffusion, cefoxitin disk diffusion and PBP2a all
performed comparably to the mecA gene test.

However, if we go to the next slide, we're looking at coagulase-negative
staphylococci. In terms of sensitivity, again, all of these tests do
very well in detecting resistance, in upwards of 98%, the oxacillin MIC,
the oxacillin disk, the cefoxitin disk and PBP2a, but let's look at the
specificity. Here you can definitely see that the oxacillin MIC and the
oxacillin disk are inferior to the cefoxitin disk or the PBP2a or mecA.
So what we're saying here is that, by using the oxacillin MIC or the
oxacillin disk for the coagulase-negative staph, we're going to overcall
resistance in a fair number of isolates. Now here, in this particular
slide, we're seeing only 94% specificity for the PBP2a. In the studies
that were done, all of these errors were basically with one species;
that being staphylococcus warneri. But the point here is that the
oxacillin MIC and the oxacillin disk diffusion test are going to
overcall resistance.

Now in the next slide, just a little bit about the recap of what we've
just gone over, because we know it's a little bit difficult to
comprehend all of these issues. Here, for staph aureus, all of the tests
work well in detecting oxacillin resistance, and the cefoxitin disk and
oxacillin disk and oxacillin MIC results are comparable. Again, for
coagulase-negative staph, the test for mecA or PBP2a work very well; but
here, too, the oxacillin MIC and also the oxacillin disk may overcall
resistance in the non-epidermidis coagulase-negative staph. The
cefoxitin disk diffusion test is better than either the oxacillin MIC or
the oxacillin disk diffusion test. For those laboratories that are doing
the oxacillin MIC test, by adding the cefoxitin disk for strains with
oxacillin-resistant MICs, this could eliminate some of the false
oxacillin resistance by increasing the specificity from 61% to 91%. So
by adding the cefoxitin disk, you can generate less false resistant
results for the coagulase-negative staph.

Now some may ask "We're not taking it up to 100% specificity and perhaps
other tests may want to be explored." Obviously the other test that
might want to be performed would be the mecA, doing the gold standard
test. We're particularly talking here about doing these supplemental
tests on isolates involved with serious infections, primarily with those
where patients would be on long-term therapy, such as endocarditis or
osteomyelitis, where it really would be important to know if that
organism is indeed truly oxacillin susceptible or oxacillin resistant.
Physicians would much prefer treating with a beta-lactam agent such as
oxacillin that the alternative, which would be vancomycin, since the
beta lactams are often more effective in treating staphylococcal
infections as compared to vancomycin. So it's important to understand
the limitations of these tests and when you might want to do
supplemental tests to better elucidate the true nature of resistance in
these particular isolates.

Now let's move on to slide 50. There's been a very brief clarification.
Here we're talking about reporting results for other beta lactams on
oxacillin-susceptible staphylococci. We've clarified this and we're now
saying that, if other beta lactams are tested and if reporting is
necessary, report results obtained. So basically what we're saying is
that, when you encounter an oxacillin-susceptible staphylococcus and
you're asked to report other beta lactams, we do not edit the results.
We actually report the actual results obtained with the in vitro test
method that we're using. This is in contrast to the rule that's been
around for years for the oxacillin-resistance staphylococci that, when
we encounter one of these, we're going to report all beta lactams as
resistant despite any susceptible result in vitro. So this is really
nothing new. We're just clarifying a little bit further how to deal with
other beta lactams when we're talking about oxacillin-susceptible
staphylococci.

Let's go on to the next slide. A question that comes up frequently is
all this stuff about oxacillin resistance and MRSA. It is necessary to
use multiple tests to confirm MRSA. There's not one hard and fast rule
that we could apply to all laboratories. Every institution has to
consider a number of factors, including technical issues. Now how
reliable is your routine test system? How often have you gotten burned
with erroneous results? How competent is your staff in working with that
particular system in your laboratory? What has been the incidence of
errors reported in the past? We have to look at our patients, the source
of the specimens. Perhaps there may be an indication in some facilities
to verify isolates from sterile body sites, but is it essential to
verify results on all wound isolates of staph aureus or presumed MRSA or
whatever? The patient history; if the patient previously was identified
as a MRSA patient, is it necessary to do supplemental testing? The
incidence of MRSA; I think a lot of us listening to this teleconference
today have been in this business, like myself, a long time and we could
recall, back in the '80s, when we saw our first MRSA. At that point in
time, we felt it was essential to confirm those results. But now with
many of our facilities seeing upwards of 50% MRSA, we have to question
whether or not we need multiple methods to confirm that we're truly
dealing with MRSA or MSSA.

Then finally, the consequences of reporting or missing MRSA, reporting
oxacillin-susceptible or oxacillin-resistant staph aureus is probably
one of the most important susceptibility results that we do report from
our laboratory. Here, too, we know, if we report a patient as having
MRSA, that patient is going to be put in isolation. That patient is
going to be subjected to broader-spectrum therapy, probably vancomycin.
That patient may be labeled as a MRSA patient and may preclude the
ability of that patient to readily be transferred to another healthcare
facility, so all of these things need to be taken into consideration
when we're deciding if we need to perform multiple tests to confirm
MRSA. I know a lot of labs out there are adding supplemental tests to
their routine system, particularly if they're using an automated system.
I personally think that this is a place that all of us need to
periodically reevaluate to see if this is the best expenditure of our
resources, because I'm personally not convinced that everyone needs to
use multiple tests to confirm MRSA in their laboratories.

Let's go on to the next slide, slide 52. Here, too, I mentioned
previously that we've grouped lugdunensis with staph aureus to the
cefoxitin disk test. We've also grouped lugdunensis with staph aureus
for MIC testing and taken it out of the coagulase-negative staph group.
Here we're seeing the same breakpoints for staph aureus and
coagulase-negative that we've seen for the past few years and there has
not been a change in the actual breakpoints listed.

Now a little bit more about lugdunensis; we don't encounter this very
often in our laboratories, but there are circumstances where it would be
really important to identify this species. I'm not an identification
person, but I did pull out some data from the Clinical Micro Procedures
Handbook and the Manual of Clinical Microbiology that has some very nice
information on the optimal tests that can be performed to identify
staphylococcus lugdunensis. Now the peculiar thing about this species is
that, as many of us know, it can be slide coagulase positive and can be
confused with staphylococcus aureus. However, in contrast to staph
aureus, it is tube coagulase negative. It is ornithine decarboxylase
positive, PYR positive and it's typically penicillin susceptible. So, on
occasion, this organism has been misidentified as staph aureus, because
of the positive slide coagulase reaction. There probably are certain
circumstances where we want to make sure that we are dealing with
lugdunensis and not a staph aureus. This would particularly be in
staphylococcal endocarditis, where it would be important for physicians
to know if it was an aureus versus lugdunensis, because lugdunensis can
be considerably more problematic that staph aureus when causing
endocarditis.

On the next slide, I've just listed some of the changes that we may see
in susceptibility results if we were to use the former interpretive
criteria for lugdunensis where it was included with coagulase-negative
staph in the M100-S14, the 2004 tables, in contrast to the recently
published M100-S15 table. As you can see, the MIC profile of lugdunensis
is usually fairly susceptible to include being susceptible to
penicillin. But a typical oxacillin result you may have encountered with
lugdunensis would have been a 0.5, which would have thrown it into the
resistant category using the old coagulase-negative staphylococcal
breakpoint. Because it was oxacillin resistant, the rule would be to
override any other beta lactam resistant, so we would have reported
those isolates as resistant to penicillin and any other beta lactam we
might have had on our panel. In this case, I've included cefazolin. In
contrast now, using the staph aureus interpretive criteria, an oxacillin
MIC at 0.5 would be reported as susceptible. We do not modify the
results for penicillin and cefazolin, so you are going to see different
interpretations using the new M100 tables, where we're now saying, "Use
the staph aureus breakpoints for oxacillin and staphylococcus
lugdunensis." We feel this better reflects for the physician those drugs
that may be used in treating these infections and we feel this is
definitely an improvement in our recommendations to you for doing
susceptibility testing and interpreting results on the lugdunensis.

Let's move on to the next slide. We'll talk a little bit about the NCCLS
interpretive criteria for vancomycin. Here I've extracted these right
from the M100-S15 document. There are really no changes in the
breakpoints for S, I and R by the MIC method. We still only have
susceptible breakpoints for the disk diffusion test. Now VISAs, or the
vanco intermediate staph aureus, have MICs at 8 to 16. The
vanco-resistant staph aureus are defined as isolates that have vanco
MICs of 32 or greater.

If you go to the next slide, I've just summarized what we know to date
about the reliability of various methods in detecting VISA or VRSA. We
know that the VISAs are not detected with the disk diffusion method. The
three VRSAs that have been encountered to date have been detected by
disk diffusion, but there may be a haze around the zone. The appearance
or the resistance may be very subtle. Here, too, it's important that, if
you're using disk diffusion testing for vancomycin in trying to identify
VRSAs, you'd have to use transmitted light and look very closely at that
zone. The reference broth microdilution MIC and agar dilution MIC and
the Etest, to this point, for those few isolates that we've encountered,
all have reliably detected the VISAs and the VRSAs. However, there have
been some problems with the automated systems in being able to identify
the VRSAs and probably the VISAs as well.

Now if you go to the next slide, this is just a recap of the three VRSAs
that have been encountered to date and the work that has been done at
the CDC. Basically the first isolate that was encountered back in 2002
had a very high vancomycin MIC of 1,024. In contrast, in the second and
third isolates that have been identified, the vancomycin MICs were
considerably lower. Unfortunately, some of the automated methods were
unable to detect these as resistant and this subsequently led to the CDC
and FDA recommending that, if you're going to be reporting vancomycin,
you can't be doing so off an automated method and that we should be
using the supplemental vancomycin agar screen test to determine whether
or not we have a VRSA strain.

In the next slide, I've included the algorithm on the CDC Web site. We
also have included the PDF version on your CD-Rom or on our Web site for
this program today. I would strongly suggest that you copy this and put
it in your procedure manual, so you would know how to react if you were
to encounter an isolate that was a potential VISA or a VRSA. It's really
important for all of us as part of our public health responsibility to
be able to identify these strains and then interact with the appropriate
public health authorities to make sure this gets reported appropriately,
because this is a very important public health issue. Here, too, I'm not
going to go over all the details of this algorithm for you. If you're
looking at a handout, there's no way you're going to be able to read any
of this, in any event, but please copy this, review it, put it in your
procedure manuals, so you will be prepared to really help with
identifying any of these isolates in your laboratory.

Now let's move on to the next slide. Here, again, I'm summarizing the
recommendations for quality assessment and quality control for the
clindamycin disk diffusion induction test, or the D zone test, and again
reiterating that, for quality control, all we need to do is to quality
control the disks either daily or weekly with our standard ATCC 25923
staph aureus. Then for quality assessment, we have the inducible strain
and the non-inducible strain that we're suggesting you use for training
your staff to assess their competency and to validate the test. So
hopefully you can appreciate the difference and, for practical purposes,
the quality assessment strains you're only going to use periodically
and, for quality control, we're going to be quality controlling the
disks each week.

Now let's move on to the next slide and this is just a chart reflecting
the changes in fluoroquinolone breakpoints for the staphylococci. On
this particular slide, I'm just showing you the MIC breakpoint changes,
but there are some corresponding disk diffusion breakpoint changes as
well. For cipro, there has not been any change. We have added
interpretive criteria or breakpoints for moxifloxacin for the first
time, but you can see that we have reduced the breakpoints for
gatifloxacin and levofloxacin.

Now if you go to the next slide, I've just listed here how are we going
to deal with these changes in the laboratory? First of all, it's
probably unlikely that all of us are going to need results from testing
these drugs against staphylococci, since they are in our supplemental
drug testing list in our NCCLS tables. The problem here, too, is that
currently our commercial systems do not have the modifications, and the
lowest concentrations on the commercial panels generally only go down to
two. So we're not going to be able to detect the resistance by using the
new breakpoints. What might be a practical strategy for dealing with
this change? Test these drugs only on physician requests and, in those
cases, use a method that will reliably detect the lower-level resistance
with the new breakpoints. The disk diffusion method will work fine using
an extended-dilution panel that goes down below the concentration of two
that will capture those lower breakpoints or to use an Etest. At this
point in time, it's been shown that it's probable that the cipro can
predict results for gati and levo, but currently we do not have this
recommendation or this algorithm in our M100 tables. If we were to
encounter an isolate that had a MIC of four or eight, either levo or
gati, we could report those as resistant.

Now let's move on and look at some of the changes with the streptococci;
only one change for pneumococci this year and that relates to the box
containing the fluoroquinolone, as shown in slide 63. Now in our former
tables, we had gati, levo and moxi in the same box connected with that
"or" connector. When we see that "or" connector, that suggests that we
can extrapolate from one drug to the other. However, it's now been shown
now that we've seen a few more quinolone-resistant pneumococci that we
cannot extrapolate from one to another. So we've removed the "or" in the
M100-S15 tables. Each of these drugs are listed individually in the box
without the "or." If we were asked to test gatifloxacin, we could not
extrapolate from levo. We would have to test each of these individually.

Now let's go on to the next slide and talk a little bit about the
changes that have occurred with testing streptococcus species. Basically
we've added the D zone test for inducible clindamycin resistance in the
beta-hemolytic streptococci. We've also included some recommendations
for when we might test the Group B strep isolated from anovaginal
screens or urine cultures in pregnant women.

Now if you'll go to the next slide, this is a little chart not unlike a
chart we showed last year during this teleconference related to
staphylococci. Basically what we're showing here is that you may
encounter erythro-resistance strains of the beta-hemolytic streptococci
that may appear susceptible or resistant to clindamycin. Now you may
have some strains that are susceptible to clindamycin due to the
presence of the "mef" gene that codes for an Efflux mechanism. These
isolates remain truly susceptible to clindamycin. In contrast, you may
have some beta strep that are susceptible to clinda-resistant erythro
due to the presence of the "erm" gene. These strains would have
inducible clindamycin resistance and it's questionable whether or not
clindamycin would be effective in these patients. Therefore, we'd have
to identify these strains to see if those strains that are
erythro-resistant, clinda-susceptible indeed includes both
clinda-resistance and true susceptibility to clindamycin. As with the
staphylococci, there are some beta strep that have the erm gene that
will constitutively demonstrate resistance to clindamycin and these are
not a problem for detection in R and D pro test systems.

Let's go on to the next slide. Although this inducible clindamycin is an
issue for all beta streptococci, I've just included Group B
streptococcus as an example. So if we were to encounter a strain that
was resistant to erythro, susceptible to clinda, the recommendation is
to not report the clinda as susceptible without performance of the D
zone test. In the next slide, I've basically used a diagram to show that
the D zone test for the beta streptococci is virtually identical to the
D zone test that we're performing for staphylococci. In the first little
photograph, you can see the recommendation for placement of the disks.
Here we're going to be using the standard Mueller-Hinton sheep blood
agar plate with your standard Kirby-Bauer, or disk diffusion test, for
the beta streptococci and placing the disks optimally 12 to 15
millimeters apart from the edge of the erythro to the edge of the clinda
disk. In the second little diagram, you can see that flattening or
truncated zone, indicating inducible clindamycin resistance. Here, too,
we would report these isolates comparable to the way we've been
reporting the staphylococci.

If you look at the next slide, this is just an example of a way you
might want to report this to call the clindamycin resistance once you've
confirmed that it does have inducible clinda resistance. But here, the
comment in our NCCLS or CLSI document is that this streptococcus is
presumed to be resistant based on detection of inducible clinda
resistance. Clinda may still be effective in some patients. So this is
still a controversial area. Particularly when you're talking about the
Group B streptococcal in pregnant women that probably haven't been
exposed to a lot of other agents, there is not good documentation that
clinda would or would not work in all of these patients. Reporting as
clinda resistant would be the conservative route, but it would be
helpful to include this comment.

Now in the next slide is a photograph of the MMWR edition that came out
in August of 2002 that deals with this whole issue of preventing
perinatal Group B streptococcal disease in pregnant women. We've
included this in a PDF file on your disk or on our Web site for this
presentation. If you've not had a copy of this in your laboratory, I
certainly encourage you to download it, print it off and review it.
There are some significant sections in there that deal with laboratory
practices, specifically talking about how to do culturing of these
specimens from the pregnant women and, also, a little bit about
antimicrobial therapy. The antimicrobial therapy recommendations
actually support the recommendations we have for testing the Group B
strep in the laboratory.

If you go to the next slide, we've included the photograph of the actual
table that suggests what drugs are recommended for using prophylactic
therapy in these moms that are colonized with the Group B strep.
Basically, just to recapture this, during the prenatal screening, the
mom is cultured to see if she has Group B streptococci in the vagina. If
so, during a vaginal delivery, she would be a prophylaxed with an agent
that would hopefully eradicate the Group B strep during delivery and
prevent the baby from coming up with Group B strep disease. As you can
see on this table right here, the recommended agents would be penicillin
or ampicillin. If the mom is penicillin allergic, but not at high risk
for anaphylaxis, cefazolin would be the alternative. If the mom is at
high risk for anaphylaxis due to a penicillin allergy, the alternatives
are clindamycin and/or erythromycin. Here, too, you can see at the
bottom of this table, if the Group B strep is resistant to clinda or
erythro or susceptibility is unknown, vancomycin would be the agent of
choice in those circumstances. Ideally vancomycin would be the last
resort if an alternative agent on this list could not be prescribed.

To support the CDC recommendations for a perinatal screening of the
Group B streptococci, we, in the M100-S15 tables, have added a
supplemental comment as indicated on slide 71. This rather extensive
comment suggests that recommendations for intrapartum prophylaxis for
Group B strep are pen or amp. While cefazolin is recommended for
pen-allergic women at low risk for anaphylaxis, those at high risk for
anaphylaxis may receive clinda or erythro. Group B strep are susceptible
to amp, pen and cefazolin, but may be resistant to clinda and/or
erythro. When a Group B strep is isolated from a pregnant woman with
severe penicillin allergy at high risk for anaphylaxis, clinda and
erythro should be tested and reported.

In the next slide, I've just indicated a way you might want to deal with
this in your laboratory. We're not suggesting you do susceptibility
testing on every Group B streptococci from prenatal screens, because
it's only a small percentage of women that are actually highly
penicillin allergic where penicillin or ampicillin cannot be used. So
you could include a comment that "Group B streptococci are susceptible
to amp, pen and cefazolin, but maybe erythro and/or clinda resistant.
Contact lab if erythro and/or clinda testing necessary." Now this is one
approach. I know some laboratories have included on their request form
under the Group B strep screen a little box to check to indicate whether
or not the mom is penicillin allergic. Then that would help the lab
identify those isolates that would be worthy of susceptibility testing.

Now let's look at the next slide. I might mention, for some unheard of
reason we can't figure out, if you downloaded your handout from the PDF
version off the Web site, you probably don't have slide 73. But
nevertheless, what this just reiterates is the incidence of resistance
to these agents we've just been talking about that might be used in
treating the Group B streptococci; basically no resistance to amp, pen
or vanco. Among the Group A, there's up to 10% erythro resistance, up to
7% clinda resistance; in the Group B, up to 25% resistance to erythro,
up to 15% resistance to clinda. These are commonly quoted rates. Select
studies have reported higher or lower rates. Nevertheless, this is
basically saying, "No resistance to amp, pen and vanco has been reported
to date, but there is some resistance to erythro and clinda in both the
Group A and Group B streptococci." I apologize for that slide not
appearing on the PDF version. I have no idea why that
happened---technology.

Let's look at slide 74, a little bit about quality assessment and
quality control and the changes that have occurred in the CLSI 2005
document. We have modified some quality control ranges. I'm not listing
those for you in this handout, but how you can identify these is that
they appear in bold-faced type in the quality control tables. We've
clarified quality control testing frequency. We've included a comment
about making sure that you follow the manufacturer's quality control
ranges if you're using a commercial system. We've expanded a little bit
in our table for which drug-bug combinations should be verified.

Now let's go to the next slide. This is just a photograph of our
Reference Guide for Quality Control Testing Frequency that has
previously appeared in the M100 tables. If you go to slide 77, we have
clarified a point that shed confusion on a lot of laboratories. This
relates to the number of days of consecutive quality control testing
required. We did say that, if the test modification was to use a new
shipment or new lot number of panel or product, only one day of quality
control testing is required. Unfortunately, some interpreted this to
mean that you no longer need to do weekly or daily quality control. That
wasn't the intent; we just were not clear in providing instructions with
this new table. So we did clarify it now to say, "This does not
eliminate the need for routine weekly or daily quality control."

Now if you go to the next slide, we've added a footnote to this
reference guide table. That is note four where we now say, "Acceptable
MIC QC limits for FDA-cleared susceptibility tests may differ slightly
from the acceptable CLSI or NCCLS QC limits." If you use a commercial
manufacturer's procedure and the quality control limits, you must follow
those quality control limits as indicated in the instructions for use.
Again, I can't emphasize strongly enough how important it is that, if
you're using a commercial product, you have to follow what is in that
product insert not only for quality control, but for every aspect of
testing.

Now let's go to the next slide. This is just showing you the expansion
to the drug-bug combinations we're suggesting be verified before you
report them on patient reports. We've added Salmonella. We're saying
that, if you were to encounter a third-generation cephalosporin or
fluoroquinolone intermediate or resistant result or a nalidixic acid
resistant result for Salmonella, you'd want to verify those results.
Also, we've added a supplemental comment. I know a lot of laboratories
aren't testing Salmonella from fecal specimens routinely, but if you are
doing susceptibility testing and you encounter a Salmonella that has
those susceptibility characteristics, intermediate or resistant to a
third-generation ceph or fluoroquinolone intermediate or resistant or
nalidixic acid resistant, communicate this to your public health
laboratory when you submit that to your laboratory. The cephalosporin
intermediate or resistant results are primarily in the Typhimurium and
the Newport serotypes.

Now if you go to the next slide, I think, for some of you that may have
downloaded from whatever version---we had multiple versions out
there---you may not see the photograph of the table for the potential
agents of bioterrorism. The only point I want to make here is just that
we have added some organisms to this table and all of this is segregated
into that table specifically for those few laboratories that might be
doing susceptible testing on the agents of bioterrorism. Again, too, we
are not suggesting any of you out there listening, with the exception of
CDC and a few other sites, to do any testing of these agents.

In the next slide, I've just listed some of the new agents that have
been introduced for the first time in our NCCLS or CLSI table in the
glossary. You can find this information in the glossary. Dalbavancin,
oritavancin, telavancin, these are newer glycopeptide drugs. If you look
at the next slide, it just says that the goal of developing these is to
have agents with improved efficacy over vancomycin and less frequent
dosing. We've also added Doripenem, a new carbapenem; Tigecycline, a
glycylcycline drug. Here, too, none of these are yet FDA cleared, but
some of them are getting close. So it's likely, in the near future,
we're going to see some of these agents in our standards and we probably
will start testing some of them as well. Next year, when we have this
teleconference, some of these may be FDA cleared and we may be testing
them.

In slide 82, I've just listed a little bit of the characteristics of
these. I've already mentioned the glycopeptides. Doripenem is similar to
meropenem. Tigecycline is a very broad-spectrum agent with the exception
of activity against pseudomonas, but it does have activity against some
of the highly resistant organisms.

Now if we go to slide 83, I just want to bring to your attention a
section of our M100 tables that might be particularly useful to you that
some of us probably never look at. This is the summary of comments and
subcommittee responses. Basically this relates to questions that some of
you may have submitted to NCCLS, and all of these get answered. Usually
the answers aren't back to you until we publish them in our NCCLS tables
the following year, but this is one way of getting some questions
answered. I know a lot of you are familiar with some of our ASM features
where you have the opportunity to ask questions as well. The summary of
those resources is listed in some of the handout information we provided
for this program.

I just want to talk, finally, about some of the issues under discussion
by the CLSI at this time. We alluded to the fact that there is much work
being done on the question of identifying ESBL producers. Also, we're
looking at the abilities of our methodologies to accurately detect all
types of beta-lactamase and other beta-lactam resistant mechanism among
the enterobacteriaceae.

In slide 83, I'm just giving you a highlight of one of the issues that
is being discussed and that is modifying the interpretive criteria or
breakpoints for some of these agents to better detect all of these
beta-lactam resistance mechanisms. So here, for example, cefotaxime, the
current susceptible breakpoint is less than or equal to eight. The
proposed or the breakpoint that's being looked at is to drop it to less
than or equal to one or less than or equal to two. So this is undergoing
a tremendous scrutiny right now and we may see some changes in the near
future in our breakpoints for the enterobacteriaceae and possibly some
other gram negatives as well, such as the pseudomonas. At this point in
time, we can't predict accurately which way this is going to go.

Slide 85, Other Issues under Discussion by NCCLS, probably by the end of
2005 or early 2006, we're going to see a new guideline for testing
bacteria not currently addressed in the CLSI or NCCLS standards to
include recommendations for susceptibility testing of Corynebacterium,
HACEK, Pasteurella, Aeromonas and so forth. There will be probably
expanded recommendations for vancomycin testing of staphylococci. We're
scrutinizing Table 1, the table that has suggestions of drugs for
testing/reporting. We're probably going to see some substantial changes
in 2006 for this; and, finally, disk diffusion testing for Neisseria
meningitidis. Again, Dr. Jorgensen's group down in San Antonio is
looking at the possibility of using a disk diffusion method for
detecting resistance of susceptibility among the meningococci.

Now on slide 86, I just want to remind you of the material we provided
with this program. In addition to the PowerPoint presentation, there's a
list of a few references and the checklist that I alluded to. I would
suggest you please look at that checklist. I think you may find it
beneficial. We've added a procedure for the BHI vanco screen for staph
aureus. We've added the MMWR Group B strep edition that I talked to you
about, the CDC algorithm for VISA and VRSA and, finally, the CLSI/NCCLS
catalog. I just want to make sure all of you are familiar with not only
the M100-S15 document we talked about today, but the large, vast number
of documents that are available from CLSI that do make life easier for
us in our laboratories.

Finally, on slide 87, I just want to acknowledge all of those
individuals that helped make this presentation possible: the Boston NLTN
office with Betsy and Shoolah and Denise; the staff at CLSI or NCCLS;
and, also, Marty Boehme, Ron Jones, Jim Jorgensen, Susan Munro, Jean
Patel, Jana Swenson, Fred Tenover, Barb Zimmer. I'm the deliverer of
this material, but an awful lot of people have played into being able to
present this information to you today and, also, the generous
educational grant from Ortho-McNeil. I know there are a lot of
Ortho-McNeil reps out there that have helped disseminate information
about this program that hopefully you found advantageous.

Slide 88 just shows the URL for the Web site, our CDC Web site, that
contains information on susceptibility testing; and, on slide 89, the
URL for the National Laboratory Training Network, who helped sponsor
this program. Again, too, I just want to remind you that, in addition to
information on susceptibility testing, the NLTN offers a variety of
programs that I think many of you may find valuable in your laboratory,
and they're usually relatively low cost.

So with that, please turn to slide 90. I know some of you might have
slide 90 labeled as "slide 900." I want to assure you that we did not go
through 900 slides today, although you may have thought that might have
been the case. I'd certainly like to thank you for your attention today.
If you have questions, please feel free to submit them. You have my
e-mail address. Be persistent if I don't answer them right away. If I
don't know the answer, I'll try to get them for you. So thank you so
very much again and for everybody who has helped make this program
possible.

D. Korzeniowski Thank you, Janet. I think we have time now for a couple
of questions. Operator.

Coordinator We have our first question coming from Chrissie DeSeco.
Ma'am, your line is open.

C. DeSeco The lab wanted to know particularly about when you were
talking about staphylococcus lugdunensis.

J. Hindler As far as identifying it?

C. DeSeco Hardly any of us have even heard of it.

J. Hindler I'm not a good identification person. Denise, are we going to
post these questions and answers on the Web site?

D. Korzeniowski We can.

J. Hindler We will send you out some information. I know some other
questions are going to come in and we would like to post the answers for
everybody to see, so I will try to get some information on that.
Probably the best thing for you to do right now---I've included in one
of those slides; there's a lot of information on that in the Clinical
Microbiology Procedure Handbook and the Manual of Clinical Micro. It'd
be best for you to review that. I'm not an expert at that and I don't
want to take the time to go over that right now.

C. DeSeco Okay, thanks.

J. Hindler We will add some information, I assure you.

Coordinator We do have our next question coming from Barbara McKee.

B. McKee We've been using VHI with vancomycin to screen our staph aureus
for several months. We've noticed there are certain strains that grow
quite well on the vancomycin agar; but when we do an Etest for
confirmation, it's got a very low MIC. Has anyone else experienced that
problem?

J. Hindler I've heard some intermittent problems about that. Again, that
work was done at CDC. What I will do is ask them to comment on that and
put that answer on the Web site as well. I don't have a lot of
experience working with that and we've not seen significant problems
here, because we've not tested it, because we're using the reference
broth microdilution system. But I will try to see if there's any
additional information that CDC might be able to shed light on that. Is
someone from CDC still on the line that might want to comment?

D. Korzeniowski Janet, I think that's all the time we have for questions
now. I apologize for the beeping; we're having a fire drill here. If
anybody's question was not answered, please e-mail Janet Hindler at
<jhindler@ucla.edu>. Again, I'd like to remind all the participants
listening into our program to register and complete an evaluation form
by February 25^th^. The directions for this are on your confirmation
letter and the handouts and we will be e-mailing the information again
to you. Documenting your participation helps us to continue to bring
high-quality, cost-effective training programs in a variety of formats.
When you've completed the registration and evaluation form, you will be
able to print your continuing education certificate. We are also
offering Florida and California CEUs. That concludes our program. The
National Laboratory Training Network would like to thank Janet Hindler
and, also, Ortho-McNeil Pharmaceutical for providing an unrestricted
educational grant. I hope that all of you will consider joining us for
future programs and that you will make the National Laboratory Training
Network your choice for laboratory training. From the State Laboratory
Institute in Boston, Massachusetts, this is Denise Korzeniowski. Good
day.

Coordinator Thank you, everyone, for joining today's conference call.
Have a good afternoon. You may disconnect your lines at this time.