In November 2005, the AGA released both a Medical Position Statement and a Technical Evaluation, which thoroughly evaluated H. pylori testing, and provided key insights on several technical points regarding H. pylori testing.  We wanted to highlight the 3 most significant recommendations as they relate to you and your laboratory.

First, the AGA now recommends that serology testing no longer be performed to test for
H. pylori because it only tests for the antibody and does not test for active H. pylori infection. This is significant as historically the majority of physicians have ordered serology for H. pylori testing, and with the new guidelines, this testing market will see a dramatic shift away from serological testing.

Second, the new AGA guidelines recommend using a Stool Antigen test, which tests for active H. pylori infection.  Because Meridian is the only company that offers  H. pylori Stool Antigen tests (Premier Platinum HpSA® Plus and ImmunoCard STAT!® HpSA), we are prepared to work with you to create a smooth transition as the serology business is converted over to stool antigen testing.

Third, the AGA now recommends that all patients presenting with Dyspepsia, who do not have alarm symptoms, have not been using NSAIDS, and who are not > 55, should be tested for H. pylori prior to being prescribed PPI medication.  Following this last guideline is important not only because it provides improved patient care, but because it also will reduce the overall healthcare costs associated with managing Dyspepsia by reducing inappropriately prescribed Rx medication, which suppresses symptoms rather than treating the underlying cause. 

As with any change, it will take time for the new guidelines to be adopted in practice, but as the laboratorian in charge of the testing, you will play an important role in educating physicians and helping to convert their orders to stool antigen testing sooner rather than later.

For more information on the AGA Guidelines or to get copies of the full AGA Guidelines, ask your local Meridian Technical Sales Representative, log on to www.hpylorilearningcenter.com, or call one of Meridian's Technical Service representatives at (800) 343-3858.

Did you know that Meridian offers a Mycoplasma test kit that is preferred by the CDC for use in children?  Originally introduced in 1994, ImmunoCard® Mycoplasma is a rapid enzyme immunoassay for the detection of IgM antibody to aid in the diagnosis of the early/acute disease state.  Mycoplasma pneumonia, also called Walking Pneumonia, causes 2 million cases of pneumonia and 100,000 hospitalizations in the US every year.  It affects people of all ages, although primary infection usually occurs in children. 

Several traditional testing methodologies are still in practice today, although dramatic improvements have been made in accuracy and diagnostic value using newer methods.  Below is a summary of several options for the detection of Mycoplasma.

• Cold Agglutination has been around for about 60 years, requires few supplies and provides fairly rapid results.  Heterophile antibodies are the target in this test creating two key shortcomings with this method of detection. Only 50% of the population produces heterophile antibodies, therefore half the positives could be missed.  Also, other viruses are known to create a similar response that would be detected by this test, yielding a non-specific positive result.  With these weaknesses, there are certainly better options.

• Culture is only about 60% sensitive and takes 2-3 weeks to return a result. By that time, the patient is likely to have passed the stage of active infection and begin to mount an IgG response.

• Complement Fixation is still commonly used and requires two sets of serum to compare the change in titers from a baseline sample.  This method does not differentiate between initial IgM and IgG response so it is a poor indication of active infection and also cross-reacts with other viruses.  Bottom line: good sensitivity, poor specificity.

• EIAs are a newer technology that offer faster turnaround time and better accuracy then traditional methods. In the microwell format results are available anywhere from 75 minutes to three hours after incubation. Rapid EIAs can reduce turnaround to just 7 minutes without any decrease in sensitivity or specificity.

ImmunoCard® Mycoplasma may not be the only rapid EIA available in the US at this time. It is however the only test that offers terrific performance, rapid time to results and a differentiated diagnosis to allow for proper and timely treatment. But don't take our word for it. Independent clinical studies from a variety of sources are easy to find. They confirm that ImmunoCard® Mycoplasma is the right choice for the detection of active infection of Mycoplasma pneumonia.

During the 2005/2006 season, you may have noticed an additional component in Binax NOW flu and RSV kits from Meridian. 

It's not a bottle of diluent or even a new procedure card.  This season Binax kits included an entry form for the Respiratory Rewards Sweepstakes. More than 8,000 entries were received by February 28, 2006 and a winner was drawn monthly from December through March.  The winners are as follows:
• December - Kim Little, Health Partners, Eden Prairie, Minn
• January -  Audie Whitaker, Community Hospital, Anderson, IN
• February - Mike McPherson, USA Women's and Children's Hospital in Mobile, AL
• March - Dr. Selvarangen, Children's Mercy Hospital in Kansas City, Missouri

These lucky winners earned a credit of $2500 towards a lab purchase with a qualified distributor. Any kit purchases of our own ImmunoCard STAT!® RSV Plus and Binax kits through Meridian qualified as did entries made on our website. 

Stay tuned for this year's upcoming Respiratory promotional programs...the season is right around the corner!

So much attention has been given recently to the idea of an Avian Influenza Pandemic, first reported in humans in 1997. After nearly a year of prolific press coverage and the promise of governmental allocations in excess of $7 billion, is there really
a threat of pandemic?

All influenza viruses have the ability to mutate, thus requiring different vaccines from year to year for seasonal influenza. So, while the avian influenza virus is not currently easily transmissible from human to human, that could change. To date, all reported human avian cases have been from poultry to human and then human to human, but no further. If the virus does mutate, sustainable human to human transfer would become more likely and a pandemic could ensue.

Since 2003, World Health Organization (WHO) reports 230 confirmed cases of human avian influenza and 130 deaths as of July 4, 2006. Of those, 181 cases and 97 deaths have occurred since the beginning of 2005. The earlier cases occurred in Southeast Asia and the Pacific Rim, where infection among poultry was well-known. More recently, human cases have been reported in Turkey and Iraq, signaling movement into Europe and the Middle East. All human cases have coincided with outbreaks of highly pathogenic avian influenza (H5N1) in birds. Avian influenza in poultry has now been identified in many European
countries and Canada as migratory birds spread the risk across land and sea barriers. Avian influenza has even been confirmed
in 4 cats in France and Germany.

As of this time, H5N1 has not been found in the US in human or avian form. If it reaches the US, it is likely that Alaska will be its’ starting
place, given the normal patterns of migratory birds from Alaska
and dispersion throughout the country.

Will a pandemic occur? It certainly is a possibility. Crossing the species barrier and reassortment of the virus would be a significant challenge necessary for it to happen. Tens of millions of poultry have been infected over three years across large geographical masses, yet the mutation to an easily transmissible human form does not yet appear to have taken place.

While no one can predict the if, how, and when of a pandemic, one thing is certain: - the world is watching closely and mobilizing plans for preventative, diagnostic and therapeutic tools to be prepared if a pandemic should occur.

Stoke Mandeville C. difficile Stool Specimen Data

Synopsis: Ten stools from the C. difficile outbreak at Stoke Mandeville Hospital in the United Kingdom were received at Meridian in December 2005. Of the 10 stools, 4 were reported to contain the 027 Outbreak Strain. This report summarizes the work done with the stools at Meridian Bioscience.

Conclusions
We were successful isolating toxigenic  C. difficile from 9/10 specimens from Stoke Mandeville (SM). Note that the UK Reference Lab isolated C. difficile from 7/10 specimens.

The UK Reference Lab reported the 027 Outbreak Strain in 4 stools. Note that Premier^TM Toxins A&B and ImmunoCard® Toxins A&B (ICTAB) were direct stool positive on all 4 of the 027 positive stools.

Rebecca Peters, David Bankert, and Arthur E. Crist, Jr.
Division of Clinical Microbiology, Department of Laboratory Services, York Hospital, York, PA 17403

Laboratory diagnosis of Clostridium difficile antibiotic-associated diarrhea or pseudomembranous colitis can be accomplished by a variety of methods (1-4). Most of the methods in routine use rely on detecting toxins (Toxin A or B) produced by the organism and excreted in the stool. Detection of C. difficile cytotoxin (Toxin B) in tissue culture is considered to be the gold standard but is labor intensive and requires 48 hours to report a negative result. Methods to detect Toxin A or A+B by enzyme immunoassay (EIA) are rapid and easy to perform but may not be as sensitive as the cytotoxin assay. The method currently in use in our laboratory to screen specimens is the C.DIFF CHEK^TM - 60, an enzyme immunoassay that detects the presence of glutamate dehydrogenase (GDH), a common antigen produced in large amounts by all toxigenic and non-toxigenic strains of C. difficile. This method is very sensitive, allowing negatives to be reported immediately after testing. However, because the test is not specific for toxin producing strains, a positive result needs to be confirmed using the specific cytotoxin assay (Toxin B) which can require an additional 24-48 hours to obtain a final result. A sensitive and specific toxin A/B assay would allow both positive and negative results to be reported immediately after testing and would assist infection control efforts to curb the spread of C. difficile within the hospital.

The purpose of this study was to compare the performance of three rapid, horizontal-flow, EIA methods to the C. difficile antigen and cytotoxin assay in routine use to determine if any of these rapid assays could replace or supplement existing assays to effectively reduce turn-around time of results.

The ImmunoCard® Toxins A & B (ICTAB; Meridian Bioscience, Inc.), Tox A/B Quik Chek (TABQC; Wampole Laboratories), X/Pect Clostridium difficile Toxin A/B (X/PECT; REMEL, Inc.), C. DIFF CHEKTM - 60 test (GDH; TechLab, Blacksburg, VA) and Cytotoxicity Assay for Clostridium difficile Toxin (CT; Bartels, Trinity Biotech Co., Carlsbad, CA) were performed according to the manufacturers instructions. The cytotoxin assay was performed on all GDH positive specimens, as well as any specimen that gave a discrepant result. All specimens were kept refrigerated and tested within 24 hours of collection.

Four hundred and ten fecal samples were evaluated. The GDH assay was positive for 104 (27%) of the specimens tested. Out of these, 62 (60%) were positive by CT giving an overall positivity rate of 15% (62/410) (TABLE 1). The sensitivity, specificity, positive predictive (PPV), and negative predictive values (NPV) of the three rapid assays compared to CT were 85%, 100%, 100%, and 97%; for ICTAB; 76%, 100%, 100%, and 96% for TABQC; and 77%, 99%, 96%, and 96% for X/PECT, respectively (TABLE 2). We did not find a correlation between a negative rapid EIA result and the time to positivity of the CT assay (TABLE 3) suggesting that factors, other than the amount of cytotoxin B present, may be responsible for false negative results. These samples have been sent for C. difficile detection by PCR but results were not available at the time of presentation.

Based on these results, we would not replace our current methods with a rapid assay since we would miss 15-24% of the CT positive specimens. However, given the fact that these assays are more rapid and less labor intensive than our current methods, one of these tests could be used in conjunction with our current testing protocol to reduce turnaround time. For example, all specimens would be initially tested by GDH followed by testing GDH positives by ICTAB. This would allow us to accurately report results on 88% (85% of the positives and 75% of the negatives) of the specimens on the same day of testing. Only 12% of our testing volume would have reports pending until results were available by the CT assay. This 3 step protocol, using list price for reagents, would increase our annual costs by $9,378. This is, however, still less expensive than performing the ICTAB assay ($80,032 per year) or CT assay alone on all the specimens.

REFERENCES
1. Doern, G.V., R.T. Coughlin, and L. Wu. 1992. Laboratory diagnosis of Clostridium difficile-associated gastrointestinal disease: comparison of a monoclonal antibody enzyme immunoassay for toxins A and B with a monoclonal antibody enzyme immunoassay for toxin A only and two cytotoxicity assays. J. Clin. Microbiol. 30: 2042-2046.
2. Merz, C.S., C. Kramer, et al. 1994. Comparison of four commercially available rapid enzyme immunoassays with cytotoxin assay for detection of Clostridium difficile toxin(s) from stool specimens. J. Clin. Microbiol. 32: 1142-1147.
3. Turgeon, D.K., et al. 2003. Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast cytotoxicity assay. J. Clin. Microbiol. 41: 667-670.
4. Wilkens, T.D. and D.M. Lyerly. 2003. Clostridium difficile testing: after 20 years, still challenging. J. Clin. Microbiol. 41: 531-534.

K. Kozak1, G. Stroup1, J. Kraft1, and A. Bubert2
1Meridian Bioscience, Inc., Cincinnati OH   2Merck KGaA, Darmstadt, Germany

ABSTRACT (Revised)
Enterohemorrhagic E. coli (EHEC) is the most common cause of hemolytic uremic syndrome (HUS) worldwide and a leading cause of acute renal failure in children. Although the majority of HUS cases have been caused by E. coli serotype O157:H7, over 200 different non-O157 EHEC serotypes have been isolated from humans. Accurate rapid tests for the detection of EHEC from both O157 and non-O157 serotypes in human stool are clearly needed. Stx1 (VT1) and Stx2 (VT2) are the principle toxins produced by EHEC. Duopath Verotoxins (Duopath, Merck KGaA, Darmstadt, Germany) was developed for the rapid detection of EHEC verotoxins (VT1 and VT2) in food and from colonies isolated from agar-based stool culture. Stool broth enrichment culture is often used for the detection of EHEC in human stool specimens. Duopath was therefore adapted for use with stool broth enrichment culture. Stool or culture isolates were grown in MacConkey Broth for 16-24 hours at 37C. Broths that showed visible turbidity were diluted with Sample Diluent Buffer developed specifically for Duopath. Diluted samples were tested simultaneously with both Duopath and an established EIA for the detection of EHEC Stx1/Stx2 in stool broth enrichment culture (Premier^TM EHEC, Meridian Bioscience). Discordant samples were resolved using agar-based stool culture. Duopath was positive with 8/9 Premier^TM EHEC positive stool broth samples (89% sensitive). The Duopath false negative sample was positive by Premier^TM EHEC near the assay cutoff (0.155, cutoff ≥ 0.150) and was agar-based culture positive for E. coli O157. Duopath was negative with 38/39 Premier^TM EHEC stool-broth negative samples (97% specific). The Duopath positive/Premier negative sample was positive by direct stool Premier^TM EHEC and yielded E. coli O157 from agar-based stool culture. Resolved sensitivity was therefore 9/10 (90%) and resolved specificity was 38/38 (100%). An overall agreement of 100% was seen between Duopath and Premier^TM EHEC with a panel of 43 EHEC and non-EHEC E. coli serotypes. Duopath Verotoxins has been adapted to provide rapid (20 minutes) and accurate results for the detection of EHEC Stx1 and Stx2 from stool broth enrichment culture.

CONCLUSIONS
The Duopath Verotoxins Test has been successfully adapted to provide rapid (20 minutes) and accurate results for the detection of EHEC Stx1 and Stx2 from GN or MacConkey stool broth enrichment culture by employing a novel Sample Diluent Buffer.

The Sample Diluent Buffer developed for the Duopath Verotoxins Test (Formulation #2) provides high specificity as well as a sensitivity that approaches
the sensitivity of the widely accepted Premier^TM EHEC EIA in stool broth enrichment culture.

J. Kraft, K. Coyle and K. Kozak
Meridian Bioscience, Inc., Cincinnati, OH

ABSTRACT
Mycoplasma pneumoniae is responsible for approximately 20% of all cases of community-acquired pneumonia. Due to the fastidious nature of the organism and the lack of commercially available antigen detection assays, serological methods have long been relied upon to help detect Mycoplasma pneumoniae infection. While IgM is a useful marker for the detection of acute Mycoplasma pneumoniae disease in children and adults under 20 years old, an IgM response is often not detectable in adults over 20 years old. Several investigators have reported the usefulness of IgA for the detection of acute Mycoplasma pneumoniae infection, especially when an IgM response is lacking. ImmunoCard STAT!® Mycoplasma IgA&IgM (ICS Myco A&M) was developed for the detection of acute Mycoplasma pneumoniae disease across all age groups. The test is a visually read recombinant antigen-based two-step immunoassay that can be completed in less than 15 minutes. A total of 52 samples (age range 1 to 80+) were tested with ICS Myco A&M and three Mycoplasma pneumoniae IgM tests (ImmunoCard® Mycoplasma, Zeus Mycoplasma IgM EIA and Zeus Mycoplasma IgM IFA).  Two out of three IgM tests had to be in agreement for a sample to be considered positive or negative for IgM against Mycoplasma pneumoniae. Discordant samples were resolved using IgA/IgM Western Blot for the P1 and P90 antigens. ICS Myco A&M was positive with 16/20 IgM positive samples (80% sensitive). Of the 4 false negative ICS Myco A&M samples, 1 was negative and 1 was equivocal by IgA/IgM Western Blot. ICS Myco A&M was negative with 26/32 IgM negative samples (81% specific). However, of the 6 false positive ICS Myco A&M samples, 4 were positive by IgA Western Blot only and 1 was positive by both IgA and IgM Western Blot. Therefore, resolved sensitivity was 21/23 (91%) and resolved specificity was 27/28 (96%). The 5 samples positive by ICS Myco A&M, positive by Western Blot and negative for Mycoplasma IgM were all from individuals at least 17 years or older. ImmunoCard STAT!® Mycoplasma IgA&IgM provides a simple, rapid and accurate method for the detection of Mycoplasma pneumoniae IgA and IgM.

RESULTS
1. IgM Reference Method Consensus
ImmunoCard® Mycoplasma, Zeus Mycoplasma IgM ELISA and Zeus Mycoplasma Antibody IgM IFA were run with a 52-member specimen panel. A specimen was considered positive when at least 2/3 IgM methods were positive. A specimen was considered negative when at least 2/3 IgM methods were negative .

2. ImmunoCard STAT!® Mycoplasma IgA&IgM vs. IgM Reference Method Consensus
ImmunoCard STAT!® Mycoplasma IgA&IgM (ICS Myco A&M) was tested with a 52-member specimen panel using the IgM reference method consensus as the gold standard (Table 1). ICS Myco A&M was positive with 16/20 IgM positive samples (80% sensitive). ICS Myco A&M was negative with 26/32 IgM negative samples (81% specific).

3. Non-Discrepant Samples by Western Blot
Western Blot was considered positive if a P1, P90 or both bands were seen with either the IgM or the IgA blots. Western Blot was considered negative when no P1 or P90 band was seen with either the IgM or the IgA blots.

A total of 14 samples that were positive by IgM Reference Method Consensus and ICS Myco A&M were run on IgM Western Blot. Nine of the samples were IgM positive for P1, P90 or both. A total of 18 samples that were negative by IgM Reference Method Consensus and ICS Myco A&M were run on IgM Western Blot. Fifteen of the samples were IgM negative for P1 and P90.

4. Resolution of Discrepant Samples by Western Blot
The 10 discrepant samples were analyzed by IgA and IgM Western Blots. Of the 4 false negative ICS Myco A&M samples, one was negative, one was equivocal by IgA/IgM Western Blot at the P1 and P90 band positions while two samples showed IgM bands at the P90 band position. Resolved  sensitivity was therefore 21/23 (91%).

Of the 6 false positive ICS Myco A&M samples, 4 were positive by IgA Western Blot only (P1 or P90 band position) and 1 was positive by both IgA (P90 band position) and IgM (P1 band position). Therefore, resolved specificity was 27/28 (96%). The 5 samples positive by ICS Myco A&M, positive by Western Blot and negative for Mycoplasma IgM were all from individuals at least 17 years or older.

DISCUSSION/CONCLUSIONS
IgM is a useful marker for the detection of acute Mycoplasma pneumoniae disease in children and in individuals under 20 years old. IgM generally appears within 1 week and usually declines within 4 weeks. However, an IgM response is often not detectable in adults over 20 years who have had multiple infections with Mycoplasma pneumoniae. Several investigators have reported the usefulness of IgA for the detection of acute Mycoplasma pneumoniae infection, especially when an IgM response is lacking. Similar to what is seen with IgM, IgA tends to rise within 1 week of the onset of Mycoplasma pneumoniae disease and generally falls within 5 weeks. The combined use of both IgA and IgM offers a reasonable approach to the detection of acute & infection across all age ranges.

No single antigen is representative or specific for acute Mycoplasma pneumoniae disease in humans. The Genzyme-Virotech Western Blot Kit describes 33 bands (antigens) that can be reactive with IgA, IgG or IgM in human serum. P1 and P90 are two Mycoplasma pneumoniae antigens that appear to be reasonably specific and are also among the most immunogenic for humans.

The ImmunoCard STAT!® Mycoplasma IgA&IgM test is based upon recombinant Mycoplasma pneumoniae P1 and P90 antigens. The Test Line yields a combined IgA and IgM result against the two recombinant antigens. Western Blot resolved data with a panel of 51 samples yielded 91% sensitivity and 96% specificity using crude-antigen based IgM reference method consensus as the gold standard. The ImmunoCard STAT!® Mycoplasma IgA&IgM test detected an IgA response in 5/6 IgM negative samples from subjects ≥ 17 years old, which were confirmed IgA positive by Western Blot.

Initial studies with ImmunoCard STAT!® Mycoplasma IgA&IgM have generated promising results for the detection of acute Mycoplasma pneumoniae disease across all age groups. Additional samples need to be tested to further validate this approach.

Many of you call into our Technical Support Hotline and speak with our wonderful team of specialists.  However, you never get the opportunity to put a name with a face - well, now you do!  Our Technical Support Staff is one of our most valuable resources at Meridian Bioscience.  In the last year, our staff answered approximately 7,000 customer inquiries and calls…that is a lot of time on the phone!  So, the next time you call into Meridian Technical Support pick up your copy of the newsletter and put a face with the name.

Meridian's Technical Support Team from left to right: Stephanie Bruns, Heather Camp, Ray Wiggins, Suzi Evans & Chris Ross.