Monday, April 08, 2013

Resistance Detection- Faster the better


Fig 1: ESBL E-test. Source
     Time and again, we take clinical microbiology seriously. If I had to say from a rough statistics, the most discussed topic in microbiology is the antibiotic resistance. I have written a few posts on the same. Needless to say, testing for resistance has been very important. A number of research has been dedicated to this field. Classic laboratory techniques, such as ESBL detection by disc synergy test are time consuming (turn around time of at least 18 hr). The most commonly suggested solution is often a molecular technique- PCR, detection of resistance enzymes by using HPLC, MALDI etc etc. Though not rocket science, the cost of molecular work, has not encouraged its use, especially in finance limited countries.

Fig 2: Pros and cons- use of molecular techniques for detecting Antibiotic resistance. 
      So the most important problem appears to be the work cost and time. Now what if I told you that, you can get your valid clinical data about antibiogram in few hours at a very low cost (When I say "Low", I mean reasonably low cost). There are many such tests and more are in development. My aim here is to introduce you to a few.

Fig 3: Carba NP test. Source
     Carbapenems, are the latest addition of wide range antibiotics, and its use is common. The commonly named drugs under this category include- Imipenem, Meropenem, Ertapenem and Doripenem. Resistance can be mediated by multiple factors, but the most common would be a carbapenemase or efflux pumping of drug. There are multiple studies that imply carbapenemase based resistance as the more prevalent cause and my experience also dictates the same. They are metallo-beta-lactamases, detected by double disc synergy, Combined disc diffusion, Hodge test or E-test. Currently there is no CLSI guidelines for the afore mentioned methods. And yes the turn around time is at least overnight.

    That brings me to the first test gaining popularity. The Carba NP Test. The test uses a simple idea. Imipenem on hydrolysis, produces a acidic condition, coupled with tazobactam and EDTA as inhibitors which can be detected using a indicator dye. That's all. The total turn around time for this test is not more than 2 hrs (in comparison to molecular tests, which takes 4-6 hrs). And what's better? It has perfect score (Sensitivity and specificity of 100%).

      Another test that uses a similar principle of acidification is the ESBL NDP (Nordmann/ Dortet/ Poirel) test. The test is based on hydrolysis of the beta -lactam ring of a cephalosporin (cefotaxime in this case), which generates a carboxyl group, by acidifying the culture medium. The acidity resulting from this hydrolysis is identified by the color change generated using a pH indicator (red phenol). The test can be done in a microwell or tube format. The technique can be applied directly to a culture plate. In a study by Nordmann et al The sensitivity and specificity was found to be 92.6% and 100%, respectively. The turn around time again is less than 2 hrs costing not more than 4 euros a test. "We can hope, in particular in many Western countries where the situation has not yet reached endemic proportions multi-resistances (France, in particular), to be able to preserve to a certain extent the efficiency of wide-spectrum cephalosporins and carbapenems, antibiotics used as a "last resource", says Patrice Normann. Source

       That brings me to the second method of analysis. Imagine this. An antibiotic is thrown at a organism. If it is sensitive, the organism is killed and degenerates. Now throw a nucleic acid specific dye (such as SYBR green I, PicoGreen, and YOYO-1). Since the cell is more permeable the dye binds to nuclear material and can be detected by using flow cytometry method. The method has been of extensive value in detection of resistance in plasmodium. Oops, but I just said, not to make use of a complicated machinery (That means no Flow cytometry). So, I recall having read a paper (For which am unable to find the reference), where you can do the same in a gel. Put some gel containing SYBR green on a slide and add anitibiotic and organism. After about 4 hours if the cell has lysed or killed, DNA will bind SYBR green and can be seen in a fluorescence microscope.

    By now, you will realize that we actually don't need too much sophisticated technology to get antibiogram results quickly. But then you would also realize that these techniques are applied to vibrant organisms (One's who are very active and replicate quickly). They are of excellent use when it comes to organisms such as Enterobacteriaceae members, Staphylocococcus etc. There are more sophisticated technologies like the eFluxx-ID screening technique (Link) and Anopore method (Link), slowly coming into practice. But, would it be of any use in say tubercle bacilli? Not much I would say.

     That brings me to the next technique. Perhaps, this is the one which has the maximum potential. Vital dyes are chemicals that can stain a cell living component. It is known that vital dyes staining correlates inversely with the multidrug resistant phenotypes. One of the old and best paper on this issue can be found here. Fluorescein diacetate (FDA) is a very well known vital dye in microbiology. It was of great use in determining variety of bacterial activity such as predicting counts. The dye was also used to assess the trend of bacterial death rather than to assess to exact number of viable bacilli (Reference).

     Let's combine the two above ideas. After treatment with a particular drug the efficacy of drug can be estimated by checking viability using FDA, say in a sputum sample. Simple. By using a little tweak the same idea could be applied in principle in the laboratory also, I would say.

Fig 4: Result of a FDA-test under the microscope: the fluorescent lines are living tuberculosis bacilli, on a background of cellular debris from human sputum. Source
       Labmedica reports (Source) as follows. "If after treatment the FDA-test was negative, in 95% of cases more elaborate tests did not find active bacilli in the patient's sputum. And if the test was positive, a resistant bacillus had been found". That summarizes whatever I want to say.

     I want to end here with a note. The use of vital dye can be extended to other bacteria testing. And non expensive techniques can be equally good in detecting resistance as the expensive molecular techniques at least in majority of the cases.
Nordmann P, Poirel L, & Dortet L (2012). Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerging infectious diseases, 18 (9), 1503-7 PMID: 22932472

Nordmann, P., Dortet, L., & Poirel, L. (2012). Rapid Detection of Extended-Spectrum- -Lactamase-Producing Enterobacteriaceae Journal of Clinical Microbiology, 50 (9), 3016-3022 DOI: 10.1128/JCM.00859-12

Johnson JD, Dennull RA, Gerena L, Lopez-Sanchez M, Roncal NE, & Waters NC (2007). Assessment and continued validation of the malaria SYBR green I-based fluorescence assay for use in malaria drug screening. Antimicrobial agents and chemotherapy, 51 (6), 1926-33 PMID: 17371812

Schramm, B., Hewison, C., Bonte, L., Jones, W., Camelique, O., Ruangweerayut, R., Swaddiwudhipong, W., & Bonnet, M. (2012). Field Evaluation of a Simple Fluorescence Method for Detection of Viable Mycobacterium tuberculosis in Sputum Specimens during Treatment Follow-Up Journal of Clinical Microbiology, 50 (8), 2788-2790 DOI: 10.1128/JCM.01232-12