Monday, May 26, 2014

Health care associated Infection- basics


     I have been recently brushing through some literature on hospital acquired infections. Needless to say, this is a matter of global concern. I have absolutely no intention of talking about the common organisms involved, but want to highlight certain basics which I believe needs a blog space.

     There are several definition that you would find on Nosocomial infections. For a reference let us consider the WHO definition. “Health care associated Infection (HCAI)” can be defined as an infection acquired in hospital by a patient who was admitted for a reason other than that infection. An infection occurring in a patient in a hospital or other health care facility in whom the infection was not present or incubating at the time of admission. This includes infections acquired in the hospital but appearing after discharge, and also occupational infections among staff of the facility.

Fig 1: Commonly encountered bacterial pathogens in HCAI.
    If you search literature on HCAI, you had see a lot of papers dealing with bacterial isolates. Irrespective of the place where the data is generated you had see the 5 organisms appearing in the important list- Acinetobacter species, Pseudomonas aeruginosa, MRSA, Enterococcus species and Klebsiella pneumoniae. If I had construct a chart of the common bacterial isolates it had look something like the one shown in Fig 1. Of these Pseudomonas aeruginosa and Acinetobacter baumanii complex, are extra ordinarily regular in showing up in culture plates. If your thinking where did the star pathogen C difficile go, I'm coming to that point of discussion in the later part.

    My first question is why Acinetobacter, Pseudomonas and MRSA are always the star Trio? The answer lies in their viability and isolation rate. It is wrong to claim that they are the common bacteria seen in the hospital. They represent a very fast growing organism in the culture plate and are viable in environment even in extreme conditions. Pseudomonas for example is one of the hardest pathogen that can be thought of. It can survive significant amount of disinfectants that are thrown on in it including phenol to an extent. That's exactly why we use Phenol agar (I used to call it Dettol agar!!!), for specific isolation. Second, they grow in the least of nutrients and easily isolated in the clinical laboratory. Hence reported. If I had consider viruses, parasites and fungus then there are much less reports available. My understanding is that Candida and Aspergillus species are more common among the fungus. Of them non albicans candida is significant.

   What causes HCAI? In the pre antiseptic era, the hospital infections where much high in number often being fatal. With introduction of Anti septic techniques this have reduced. But the single most important thing among them is Hand-washing. As more recent papers indicate, there is still less than 40% compliance in health care workers. This is the most important factor even to this day. Solution to the problem exists such as use of Hand-rubs but much less of it is practised than said.

    A question that I once thought of was "Shouldn't it be that only organisms originating from health care set up be included in hospital acquired infections?". Let me elaborate. If a patient is put on a urinary catheter, and the patient acquires E coli- UTI, should it be called Hospital acquired. The organism came from the patient himself. The answer is still "Yes". It doesn't matter where did the organism actually come from. The essence is "If its a product of medical intervention?".

   Despite a century old history of Nosocomial infections, there is a little data on the dynamic of these infections. The first step to study the actual dynamics of such a complicated system involving multiple variables is to know what the hospital actually harbours. This requires an extensive analysis. A simple petri dish culture involves only a few organisms. Modern third generation sequencers have enabled us to look into much more depth. In other words, "Hospital Microbiome" is the data we are now looking for. There currently is a project going on (Link), with some preliminary data.

    There has been some studies trying to get the hospital system more resistant to harbouring pathogens. One of the most well known approach include use of bacteriophage solutions that maybe sprayed on surfaces. The approach though fascinating, is not a long time viable approach. More recently the concept of use of copper surfaces in the hospital has shown promise.
     In short, hospital acquired infections represent a standard problem of modern day health care system. The problem is currently addressed by frequent surveillance, constant vigilance of Infection control committee and safe medical practices. However, it is also true that hospital is a microbial heaven.
Smith, Daniel. (2013-04-15) The Hospital Microbiome Project: Meeting Report for the 1st Hospital Microbiome Project Workshop on sampling design and building science measurements, Chicago, USA, June 7th-8th 2012. , 8(1), 112-117. DOI: 10.4056/sigs.3717348

Allegranzi B, Bagheri Nejad S, Combescure C, Graafmans W, Attar H, Donaldson L, & Pittet D (2011). Burden of endemic health-care-associated infection in developing countries: systematic review and meta-analysis. Lancet, 377 (9761), 228-41 PMID: 21146207

Saturday, May 10, 2014

Does Toxoplasma drive you crazy?


      A couple of times I have talked about if a microbe can influence the Neurochemistry. I have made my points clear as this can be possible in my previous post. I got a couple of comments as to why I have not talked about Toxoplasmosis in this context. So let me elaborate on the issue and consider this as a sequel to my previous post on schizophrenia (Read my previous post here). There has been a lot of debate on this issue and press releases have sometimes hyped the scene. There are facts known in the science world, that serve as examples of how one species can entirely manipulate the other as demonstrated by Ed Yong in a TED talk (Link).

The following paragraph was published in a magazine the Week (Original Source; Link) published in 2012

"Scientists are still trying to understand the behavioral changes - if any - in humans infected with toxoplasma, but research has so far linked it to worse reaction times, a more than two-fold increase in the risk of being involved in a car accident, suicide in women and schizophrenia".

      I usually say this sentence to begin with in my lectures. "Toxoplasma is a kind of marvel pathogen. It can infect everything which is warm blooded (so goes the saying) and it can be as silent as non existent in a system".

Fig 1: Ultra structure of Toxoplasma.
    Basically an apicomplexan, related to Plasmodium species has been well studied over the years. Despite extensive molecular evolutionary work, it seems that there is very less clonal lineages and probably the only species in the genus. As I said, it can infect almost anything which is warm blooded. But for discussion purposes, the natural host is considered to cycle between the cat and the rat. There is an excellent review by Boothroyd and Black in MMBR, about the structure, life cycle and molecular aspects of toxoplasma which I recommend reading to get a very detailed understanding of the subject. Link

    Here's an important point. In the life cycle asexual part of development happens in the mammals. In this case, mice infected with toxoplasma. It so happens that mice looses the fear of cats and will wander in its territory allowing the cat to grab the mice. And the parasite gets to continue its life cycle in the cat which repeats. The phenomenon is the same in wild, where the felines and mammals form part of the cycle. It was thought that this suicidal behavior is what is seen in humans having latent toxoplasma infection. Or is it?

     Let's get a little deeper. What is the mechanism of mice loosing the fear of cat. Mice that have been infected with toxoplasma, will loose the fear of the cat, and this behavior persists even after the infection is gone for months. Its a permanent change. As Eisen puts it (Source) "Long after we lose the ability to see it in the brain, we still see its behavioral effect". This is because of the following. Prey in general, detect the presence of a hunter by smell especially from urine. This is achieved by olfactory recpetors called as trace amine-associated receptors (TAARs). It is proposed that the receptors are somehow damaged of their effects. No smell, no fear. This is a very clean explanation. From evolutionary point of view, it is important for the parasite since continuation of its sexual life cycle requires that it move to the felines.

   But what about we humans? We don't depend too much on our smell to recognize situation. So what causes the human behavioral change. A good deal of possibility is that parasite in brain can elicit immune response, and certain types of immune response especially certain interleukin can influence behavior change. This is a subject of investigation in the field of psychoneuroimmunology of schizophrenia. But there appears to be a growing evidence of second possibility.

Fig 2: Dopamine hypothesis of schizophrenia. Source
     Micro RNA or in short miRNA are short stretches of non coding RNA of about 22 nt in length. They are an integral part of regulation in the RNA world, and are usually involved in fine tuning of cellular machinery. a cyclic AMP-responsive element binding (CREB)-regulated miRNA. miR-132 is known to have a regulatory role in the dopmaine regulation. A recent publication has shown that toxoplasma strains consistently up regulated the miR-132 and is associated with changes in dopamine receptor signaling. That calls for some attention, since schizophrenia has a known link with miR132. As a matter of fact there are 2 hypothesis for schizophrenia- Glutamate and Dopamine hypothesis. The dopamine hypothesis is depicted in Fig 2.

       This concept raises an important question. How does toxoplasma gain anything from an evolutionary perspective. In other words, why does toxoplasma still have this trait if at all the explanation is true for schizophrenia. The answer probably lies that we are asking the wrong question. As Vincent explains (Link) in his blog, virulence maybe a parallel property and not directly related to life cycle. The pathogen has it cause it is a side effect of some mechanism rather than toxoplasma itself is interested in people having suicidal tendencies and schizophrenia. So where does the collateral explanation for dopamine comes from? It should be noted that the toxoplasma has two genes for the enzyme tyrosine hydroxylase, the key enzyme for synthesis of dopamine, and bradyzoites express these enzymes and release dopamine into surrounding tissue. Dopamine release into surrounding tissue is important for toxoplasma, probably by helping it to progress with tachyzoite production, as suggested by Strobl etal.

   As per the literature, I can sum up few possibilities of how a pathogen can get your neurochemitsry to work different. First organisms can directly interfere with signalling mechanisms such as toxoplasma effects on mir-132, Gut flora interfering with microbiota–gut–brain axis etc. Second, the organism may illicit a cross reactive antibody such as in the case of Group-A Beta-Hemolytic Streptococcus pyogenes causing ABGA (Anti basal ganglia antibodies) or production of cytokines which impacts through hypothalamic-pituitary-adrenal axis which is the seat of integrating endocrine, immune and cytokine pathways with neural signalling.
Ingram WM, Goodrich LM, Robey EA, & Eisen MB (2013). Mice infected with low-virulence strains of Toxoplasma gondii lose their innate aversion to cat urine, even after extensive parasite clearance. PloS one, 8 (9) PMID:24058668

Flegr J (2013). Influence of latent Toxoplasma infection on human personality, physiology and morphology: pros and cons of the Toxoplasma-human model in studying the manipulation hypothesis. The Journal of experimental biology, 216 (Pt 1), 127-33 PMID:23225875

Xiao J, Li Y, Prandovszky E, Karuppagounder SS, Talbot CC Jr, Dawson VL, Dawson TM, & Yolken RH (2014). MicroRNA-132 dysregulation in Toxoplasma gondii infection has implications for dopamine signaling pathway. Neuroscience, 268, 128-38 PMID: 24657774

Flegr J (2013). How and why Toxoplasma makes us crazy. Trends in parasitology, 29 (4), 156-63 PMID: 23433494

Strobl JS, Goodwin DG, Rzigalinski BA, & Lindsay DS (2012). Dopamine stimulates propagation of Toxoplasma gondii tachyzoites in human fibroblast and primary neonatal rat astrocyte cell cultures. The Journal of parasitology, 98 (6), 1296-9 PMID: 22512377

Cryan JF, & Dinan TG (2012). Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour. Nature reviews Neuroscience, 13 (10), 701-12 PMID: 22968153

Thursday, May 08, 2014

Combo Therapy for TB


      Talking about tuberculosis, its a hard pathogen to beat. The tough part of the problem is that it is a very sluggish bacteria. It is indeed very difficult for antibiotic drugs to be designed. But scientists have come out with breakthroughs. There is a great deal of effort in improving the vaccines, exploring new drugs (See my previous posts here and here). But probably there is another part of the problem. TB, at least in the developing world, is well associated with other parallel infectious conditions especially HIV. That makes a combo drug very attractive candidate to work on.

     A trio drug combination referred as PaMZ is making some headlines. The short form stands for 3 drugs PA-824, moxifloxacin and pyrazinamide. Moxifloxacin is a well known drug candidate to treat MDR TB. It is a fourth-generation synthetic fluoroquinolone antibacterial agent, which kills by inhibiting DNA gyrase. Pyrazinmaide is a known drug used in first line treatment of TB. Out of the 3, the 2 drugs are well known with their function and capabilities.

      During the preparation of bicyclic nitroimidazofurans, which were investigated as radiosensitizers for use in cancer chemotherapy, Nitroimidazo oxazoles was incidentally, found to possess activity against cultured replicating Mycobacterium tuberculosis. Based on the findings, several derivatives were created. Testing the derivatives showed several compounds with high and specific activity against TB. PA-824 was one of the candidates in the original NAP series. It was not in the competing top list, compared to other derivatives, but proved to be the best when studied using a animal model. The lead compound was CGI-17341, which was not further developed due to its mutagenic activity.

Fig 1: PA-824 activity. Source
      Studies, by comparing it with metronidazole showed a promising activity. Metronidazole was used for comparisons because, it was one of the first candidates to have known activity against latent TB, and PA-824 belongs to same superclass of chemical. The complete name of PA-824 is (S)-2-nitro-6-[4-(trifluoromethoxy)benzyloxy]-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine, was identified as the lead 4-nitroimidazo-oxazine. The mechanism of action appears to be via release of RNS (reactive nitrogen species). The drug by itself is inactive and activated by a deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis (It catalyzes the reduction of multiple nitroimidazoles through F420-dependent nitroreduction). In 2002 the Global Alliance for TB Drug Development licensed PA-824 and related nitroimidazole compounds for further development.

          The combination of the 3 drugs which was expected to produce faster action comes from the ability of PA-824 and Pyrazinamide to attack latent cells and the efficacy of Moxifloxacin, which is a well known drug for MDR TB. The drug successfully tested in phase 1 and phase 2 trials, which was recently completed. All the hype is about the drug entering phase 3 with high quality results expected. There are expectations as to some other combinations will also work. Some of them are currently in clinical trials. This includes- JPaZ which stands or bedaquiline (J), PA-824 (Pa) and pyrazinamide (Z), which is soon to enter phase 2b (Link). Independently tested drugs such as Clofazimine has shown promising activity and planned to be tested in combinations with other drugs.

      CGI-17341 as I mentioned, was a lead compound, but had mutagenic activity. Otsuka Pharmaceutical Co. Ltd, overcame the mutagenicity problem by substituting the 2-position of the side chain which led to the compound OPC-67683. The compound is currently in a Phase 2 study.

     The potential high market for antibiotics against TB is leaving a trail of research. In next few years, we will have a lot more Anti-TB compounds, for good
Cellitti SE, Shaffer J, Jones DH, Mukherjee T, Gurumurthy M, Bursulaya B, Boshoff HI, Choi I, Nayyar A, Lee YS, Cherian J, Niyomrattanakit P, Dick T, Manjunatha UH, Barry CE 3rd, Spraggon G, & Geierstanger BH (2012). Structure of Ddn, the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis involved in bioreductive activation of PA-824. Structure, 20 (1), 101-12 PMID: 22244759

Mukherjee T, & Boshoff H (2011). Nitroimidazoles for the treatment of TB: past, present and future. Future medicinal chemistry, 3 (11), 1427-54 PMID: 21879846