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PulseNet Enteric Pathogens

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  1. ENTERIC BACTERIAL INFECTIONS PulseNet Current Applications Dr.T.V.Rao MD Dr.T.V.Rao MD

  2. Enteric Pathogens – Emerging Trends • The emerging trends in Enteric pathogens both in spread and Antibiotic resistance is a concern to the Medical Profession, and Community at Large, The problems much bigger in Developing world where it can carry higher morbidity. The Microbiologists should play crucial with help of community health workers. Dr.T.V.Rao MD

  3. Important Organisms • Norwalk-like viruses • Campylobacter • Salmonella • E. coli O157:H7 • Clostridium botulinum • Shigella spp • Toxoplasma • Emerging organisms Dr.T.V.Rao MD

  4. Enteric Pathogens • Enteric Pathogens are gastrointestinal organisms spread by contamination of foods mainly of animal origin and among people who may be carriers. • Global, regional and national estimates clearly place diarrhoeal diseases as a major, albeit a substantially neglected, public health problem. Deaths of children aged <5 years owing to diarrhoea was estimated to be 1.87 million at the global level (uncertainty range from 1.56 to 2.19 million), which is approximately 19% of total child deaths Dr.T.V.Rao MD

  5. Majority Present as Gastro-Enteritis • Gastro-enteritis is a major public health problem in the world especially among developing countries. • Diarrhea is one of the leading causes of mortality and morbidity in developing countries • An estimated 1000 million diarrheic episodes (2.6 episodes per child per year) and 3.3 million deaths per year (range, 1.5-5.1 million per year) occur each year in children < 5 yr of age globally Dr.T.V.Rao MD

  6. Epidemiology • Many unrecognized or unreported • Mild disease undetected • Same pathogens in water and person to person • Emerging pathogens unidentifiable • Greatest risk • Elderly • Children • Immunocompromised Dr.T.V.Rao MD

  7. Diarrhea can be significant Presenting illness • Diarrhoea is a syndrome that can be caused by different bacterial, viral and parasitic pathogens. Accurate understanding of the cause of diarrhoea in a given setting is an onerous task that requires systematic monitoring of the various pathogens. The availability of a well equipped clinical microbiology laboratory is a prerequisite to undertake such studies. Dr.T.V.Rao MD

  8. Main Etiological Microbes • The main etiology of the diarrhea is related to a wide range of bacteria (such as, Shigella spp., Salmonella spp., E. coli and Vibrio cholerae) entero-parasites (Giardia spp. and Entamoeba histolytica), and viruses (Rotavirus, Adenovirus and Norwalk virus) . In industrialized countries rotavirus predominates and bacteria are commonly found in children from developing countries Dr.T.V.Rao MD

  9. Routine culturing of fecal Specimens …. • However, the laboratory investigation of faecal samples from a patient suffering from gastroenteritis usually focuses on the detection of Campylobacter spp., Salmonella enterica, Shigella spp. and verotoxigenic Escherichia coli, which, except for shigella, can zoonotic. Dr.T.V.Rao MD

  10. Major Scientific Studies Identified • The most frequently isolated of these pathogens is campylobacter, primarily C. jejuni subsp. jejuni (other species include C. coli, C. upsaliensis, C. hyointestinalis and C. fetus). All of these species, except for C. fetus, grow best between 37 and 43ºC. Most infections are sporadic, though outbreaks can occur, and the most common sources of infection include poultry, water and milk [1]. Dr.T.V.Rao MD

  11. MacConkey agar differentiates Major Pathogens Dr.T.V.Rao MD

  12. MacConkey agarContinues to be Minimal essential Media to Identify Enteric Pathogens Dr.T.V.Rao MD

  13. Specimens were cultured …. • The samples were cultured using standard microbiology techniques to observe Salmonella-Shigella and V. cholerae colonies • Antibiotic sensitivity was done by Kirby Bauer disc diffusion method using Diagnostic Sensitivity • Follow as per the recommendation of National Committee for Clinical Laboratory Standards • (NCCLS) . Or CLSI guidelines Dr.T.V.Rao MD

  14. Choose the Optimal Antimicrobial Agents • All the common isolates are tested with the following antibiotic discs; trimethoprim/ sulphamethoxazole (co-trimoxazole), ampicillin sulphamethoxazole, tetracycline, erythromycin, chloramphenicol, amoxicillin/clavulanic acid, cephalothin, nalidixic acid, gentamycin, cefuroxime, cefotaxime, ciprofloxacin, ceftazidime, ceftrioxone. The results were recorded as either sensitive or resistant in this study. Dr.T.V.Rao MD

  15. Document results with WHONET software • WHONET is a free Windows-based database software developed for the management and analysis of microbiology laboratory data with a special focus on the analysis of antimicrobial susceptibility test results. Dr.T.V.Rao MD

  16. WHONET Helps to keep track of Antibiograms • To enhance the local use of data for local needs: clinical decision support, antimicrobial use policy, infection control and outbreak detection, identifying laboratory test performance, and characterization of local microbial and resistance epidemiology To promote local, national, regional, and global collaborations through the exchange of data and sharing of experience Dr.T.V.Rao MD

  17. E.Coli not just Commensal but Emerging Importance • Diarrhoeagenic Escherichia coli is categorized into the followingsix pathotypes: enteropathogenic E. coli (EPEC), enterotoxigenicE. coli (ETEC), enterohaemorrhagic E. coli (EHEC), enteroinvasiveE. coli (EIEC), diffusely adherent E. coli (DAEC), and enteroaggregativeE. coli (EAEC). Other diarrhoeagenic E. coli pathotypes havebeen proposed, such as cell detaching E. coli (CDEC); however,their significance remains uncertain (Abduch-Fabrega et al., 2002;Clarke, 2001 Dr.T.V.Rao MD

  18. E.Coli are a Growing Concern Dr.T.V.Rao MD

  19. Diarrhoeagenic E.coli emerging pathogen . Each of the pathotypes has distinguishing characteristicsrelated to epidemiology, pathogenesis, clinical manifestationsand treatment. EAEC is the most recently identified and describeddiarrhoeagenic E. coli. This bacterium was described in 1987,and identified in a child from Chile with persistent diarrhoea(Nataro, 2005). Dr.T.V.Rao MD

  20. E.coli can produce serious disorders • EAEC is increasingly recognized as an emerging enteric pathogen.EAEC is a cause of persistent diarrhoea and malnutrition inchildren and HIV-infected persons living in developed countries,is the second most common cause of travelers' diarrhoea (ETECis the most common cause), and is a common cause of acute diarrhoealillness in children and adults (4.5 %) presenting to emergencydepartments and inpatient units in the USA (Cohen et al., 2005;Nataro et al., 2006). Dr.T.V.Rao MD

  21. EAEC can be A bioterrorism agent • EAEC is also considered a potential bioterrorismagent (National Institutes of Health category B) (Huang & DuPont, 2004;Huang et al., 2004b). The objective of this review is to providean update on this increasingly recognized emerging enteric pathogen Dr.T.V.Rao MD

  22. Identification of EAEC • The gold standard for identifying EAEC is the HEp-2 cell-adherenceassay (Nataro & Kaper, 1998). This assay identifies EAECby its ‘stacked brick’ aggregating phenotype (Fig. 1).Variations in the assay have been described. A formalin-fixedHEp-2 cell-adherence assay is reported to be sensitive (98 %)and specific (100 %) compared to the traditional assay, whilereducing the risk of contamination (Miqdady et al.,) 2002only inresearch settings, and is labour intensive (Huang et al., 2004b).A clump formation test has also been described to be usefulin the identification of EAEC (Iwanaga et al., 2002). Dr.T.V.Rao MD

  23. HEp-2 cell-adherence assay of EAEC, showing the aggregative ‘stacked-brick’ pattern. Dr.T.V.Rao MD

  24. Salmonella can cross the Barrier of Intestines • Invasive enteric pathogens of the Salmonella or Shigella genera initiate infections by invading the intestinal epithelium. Depending on the species, salmonellae either translocate across the mucosa of the small intestine and cause a systemic febrile disease or they evoke a localized inflammatory response in discreet areas of the infected mucosa. Dr.T.V.Rao MD

  25. Shigellae infections • Shigellae infections is characterized by gastroenteritis, and a choleragen-like enterotoxin may contribute to the symptomology. Shigellae can also evoke diarrheal episodes; however, classic shigellosis is characterized by localized invasion of the colonic epithelium, with inflammation and ulceration of the mucosa. Derangement of the colonic mucosa is manifested in the bloody, mucoid stool characteristic of bacillary dysentery Dr.T.V.Rao MD

  26. Campylobacter jejuni An important food borne Infection • Campylobacter jejuni is a common cause of entero-invasive diarrhea in man. The disease is often milder than that caused by Shigellae. This reflects the specific exposures of the age group and eating habits. The majority of infections are sporadic, although occasionally outbreak associated, particularly with hygiene breaches in the preparing or serving poultry. Outbreaks of campylobacter's enteritis have been associated with contaminated water and raw milk. Usually diarrhea due to campylobacter's is self-limiting and does not require therapy unless the individual is immunosuppressed or the infection is extra-intestinal. Dr.T.V.Rao MD

  27. Genus Salmonella • The genus Salmonella comprises two species, namely S. enterica and S. bongori, which have more than 2,500 serotypes or serovars, differentiated on the basis of their somatic (O) and flagellar (H) antigens by the White-Kauffmann-Le Minor Scheme[2]. Most salmonella infections are confined to the gastrointestinal tract. Dr.T.V.Rao MD

  28. Genus Shigella • here are four species in the genus Shigella; Sh. sonnei (1 serotype), Sh. flexneri (14 serotypes), Sh. boydi (18 serotypes) and Sh. dysenteriae (13 serotypes), all of which are characterised serologically on the basis of the O antigen only, as Shigella spp. lack the H antigen [3]. Shigella dysentery is confined to humans and primates, and transmission, which is most common in developing countries, is human-to-human or via contaminated water or food. Dr.T.V.Rao MD

  29. E. coli • Strains of E. coli that produce a protein toxic to Vero cells are termed verotoxigenic E. coli (VTEC), and are capable of causing two types of disease, namely haemorrhagic colitis and haemolytic uraemic syndrome [4]. Serogroup O157 is the most common cause of these illnesses, but at least 150 serotypes have been identified as verotoxigenic [5]. The two toxins, VT1 and VT2, were also termed “shiga-like”, or “STEC”, due to their similarity to the toxin of Sh. dysenteriae [5]. Dr.T.V.Rao MD

  30. Multiplex systems identifies Multiple organism • To date, only one commercial multiplex system is available for the detection of the four pathogens. This is the EntericBio system (Serosep Ltd., Limerick, Ireland). It combines overnight broth enrichment with PCR amplification and detection by hybridisation [Figure 1]; results can be available within 32 hours of receipt of a specimen in the laboratory. Dr.T.V.Rao MD

  31. Antibiotics have changed the Medicine • One of the greatest accomplishments of modern medicine has been the development of antibiotics for the treatment of potentially fatal infections. However, this has inevitably been followed by the acquisition of resistance towards their antimicrobial activity. Dr.T.V.Rao MD

  32. Appropriate Antibiograms help the Clinicians • It is important to bear in mind that the principal purpose of monitoring antibiotic resistance trends among enteric pathogens is to provide clinicians with data that can be used to select appropriate treatment regimens. Antimicrobial testing should include antibiotics that are currently being used for the treatment of bacterial diarrheas i.e. fluoroquinolones and azithromycin. Dr.T.V.Rao MD

  33. Emerging Drug Resistance • About 70 percent of the bacteria that cause infections in hospitals are resistant to at least one of the drugs most commonly used for treatment. Some organisms are resistant to all approved antibiotics and can only be treated with experimental and potentially toxic drugs. An alarming increase in resistance of bacteria that cause community acquired infections has also been documented, Dr.T.V.Rao MD

  34. Drug Resistance a Growing Concern • In the past two decades have seen a marked decline in the discovery and development of novel antibiotics and a remarkable increase in resistance to those currently available.1 In particular, there is substantial concern worldwide with the mounting prevalence of infections caused by multidrug-resistant Gram-negative bacteria, in particular Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae; for these species, polymyxins are sometimes the only available active antibiotics. Dr.T.V.Rao MD

  35. The continuing need for culture of bacterial enteric pathogens • For epidemiological purposes, the identification of a salmonella strain to serovar level is used universally, and the subtyping of a serovar is often performed, using bacteriophage analysis or a variety of molecular-based typing methods after DNA extraction from culture. Shigella isolates are speciated and typed using antisera, and may be subtyped. A confirmed E. coli O157 isolate is commonly phage typed. These characterisation tests require culture, as does most antimicrobial susceptibility testing. Dr.T.V.Rao MD

  36. Genetics and Channing Taxonomy • Understanding the potential plasticity of bacterial genomes, it may be necessary when using molecular methods for diagnosis, to consult the international databases regularly for evidence of changing genetics and updated taxonomy. Dr.T.V.Rao MD

  37. Molecular Biology • Molecular biology involves the study of macromolecules (DNA, RNA, proteins) and the macromolecular mechanisms found in living things, such as the molecular nature of the gene and its mechanisms of gene replication, mutation, and expression. Dr.T.V.Rao MD

  38. Molecular Biology • In the context of infectious disease epidemiology, the molecular biologic approach involves molecular characterization of disease –causing organisms and their subdivision by their DNA, RNA and/or proteins. • DNA “fingerprinting” • Sub typing • Molecular sub typing Dr.T.V.Rao MD

  39. Molecular Epidemiology • Molecular Epidemiology = Epidemiology of disease in affected population + Molecular Characterization (sub typing) of Etiologic Agent Dr.T.V.Rao MD

  40. Networking with PulseNet • PulseNet is a national network of public health and food regulatory agency laboratories coordinated by the Centers for Disease Control and Prevention (CDC). The network consists of: state health departments, local health departments, and federal agencies (CDC, USDA/FSIS, FDA). Dr.T.V.Rao MD

  41. What is PulseNet • A key tool in recognizing and controlling food-borne outbreaks is high-discriminatory molecular typing of the pathogen isolated from humans and their possible sources. Because strains from suspected outbreaks, which may be isolated over geographical and temporal distances, require urgent investigation, a strategy involving local DNA fingerprinting of such strains and electronic submission and analysis of the results to a central database system will be implemented. Dr.T.V.Rao MD

  42. PulseNet is a integrated Network connecting Molecular reports • PulseNet participants perform standardized molecular sub typing (or “fingerprinting”) of food borne disease-causing bacteria by pulsed-field gel electrophoresis (PFGE). PFGE can be used to distinguish strains of organisms such as Escherichia coli O157:H7, Salmonella, Shigella, Listeria, or Campylobacter at the DNA level. DNA “fingerprints,” or patterns, are submitted electronically to a dynamic database at the CDC. These databases are available on-demand to participants—this allows for rapid comparison of the patterns. Dr.T.V.Rao MD

  43. How the PulseNet works • The overall aim is to establish a real-time linked surveillance database system to detect disease clusters and investigate outbreaks of Salmonella, VTEC and Listeria. The participants of the network will perform standardized molecular sub typing of the involved organisms and submit them to the database system following a strict Quality Assurance system. Medical and veterinary curators for each organism will be responsible for the daily function of the databases including cluster detection and response. Dr.T.V.Rao MD

  44. Pulsed Field Gel Electrophoresis (PFGE) • DNA macro restriction analysis utilizes restriction enzymes that cut genomic DNA infrequently and thus generates a small number (usually 10-20) of restriction fragments. These fragments are usually too large to separate by conventional agarose gel electrophoresis. However, these fragments can be effectively resolved by a process termed pulsed-field gel electrophoresis (PFGE), developed in 1984 to separate yeast chromosome-sized DNAs Dr.T.V.Rao MD

  45. Pulsed Field Gel Electrophoresis (PFGE) • PFGE facilitates the differential migration of large DNA fragments through agarose gels by constantly changing the direction of the electrical field during electrophoresis. The contour-clamped homogeneous electric field (CHEF) gel electrophoresis method has become the method of choice for resolving DNA macrorestriction fragments of bacterial genomic DNA. Dr.T.V.Rao MD

  46. Global Networking with PulseNet • These independent networks work together in PulseNet International allowing public health officials and laboratories to share molecular epidemiologic information in real-time and enabling rapid recognition and investigation of multi-national food borne disease outbreaks. Routine communication between the various international PulseNet networks will provide early warning on food borne disease outbreaks to participating public health institutions and countries Dr.T.V.Rao MD

  47. Dr.T.V.Rao MD

  48. PulseNet Participants - PulseNet Asia-Pacific • AustraliaMalaysia Bangladesh New Zealand China Philippines Hong Kong Taiwan India Thailand Japan Vietnam Korea Dr.T.V.Rao MD

  49. India Host for PulseNet Asia-Pacific Meeting 2008 Dr.T.V.Rao MD

  50. PulseNet USA, Connects Molecular Epidemiology • PulseNet USA, the national molecular sub typing network for food borne disease surveillance, began functioning in the United States in 1996 and soon established itself as a critical early warning system for food borne disease outbreaks, particularly those in which cases may be geographically dispersed. The PulseNet network is now being replicated in different ways in Canada, Europe, the Asia Pacific region, and Latin America. Dr.T.V.Rao MD