EMERGING CHALLENGES IN DIAGNOSTIC MICROBIOLOGY

NEED FOR NEWER TECHNOLOGIES Dr.T.V.Rao MD

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•Delivered as Guest Lecture as part of the Dr K RajyaLakshmi oration at SVS

Medical College, Mahabubnagar –Telangana on the occasion of XIX th

Annual conference IAMM TS AP State Chapter

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A Tribute to my Teachers Who Taught me Microbiology

• I am grateful to my Teachers

Dr ( Prof) B Raja Rao Garu

Dr ( Prof) Late Saroja Venugopal Madam

Dr ( Prof ) Ramani Madam

Dr ( Prof) Joga Lakshmi Garu

Dr (Prof ) Ramanamma Garu

Many others Who Taught me Microbiology

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Microbiology Moving from Past to Future

•The long span of four hundred and fifty years of microbiology has brought amazing insight into the biology of microorganisms and has also brought with it new challenges, and close involvement with clinical staff in a variety of acute and community settings to effectively manage infections

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Microbiology Moving from

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Beginning of Diagnostic Microbiology

•In the late 1800s, the realization that identifiable microbes caused specific diseases led to pathogens Specific medical diagnosis. Although the time honoured techniques of growing bacteria in broth or solid cultures and staining and examining them under microscopes are still important today01-02-2016 Dr.T.V.Rao MD 6

The real Good of the Past in Diagnostic Microbiology

• In the good old days, the microbiology laboratory used to be a labour intensive place equipped with incubators and microscopes. Microbiologists were patient scientists waiting at least 24 hours before their isolated cultures were grown enough for identification

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Laboratories are Evolving • Today, laboratory medicine

is developing at a rapid pace and the microbiology lab is having its own evolution going on. Lab automation is emerging and processes are done faster than ever with more standardized and comparable tests.

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Microbiology in 21st Century

• In the twenty-first century, the clinical microbiology laboratory plays a central part in optimizing the management of infectious diseases and surveying local and global epidemiology.

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Where we stand Today• Most neglected and least

invested specialty• Reasons can be many

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Newer Adoptions in Microbiology

•This pivotal role is made possible by the adoption of Rational sampling, point-of-care tests, extended automation and new technologies, including mass spectrometry for colony identification, real-time genomics for isolate characterization, and versatile and permissive culture systems

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SPECIMEN COLLECTIONwhose duty it is ?

The specimen is the beginning. Alldiagnostic information from the laboratorydepends upon the knowledge by whichspecimens are chosen and the care withwhich they are collected and transported.

—Cynthia A. Needham

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A Simple question to Many Microbiology Friends

How many Microbes we are identifying How Good we are in Conventional Methods

to identify simple non fastidious Microbes

How many really measuring the Zone sizes accurately when we are reporting Sensitivity and resistance patters as per CLSI guidelines

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What the Hospitals and Administrators expect from Us

•Today’s microbiology labs are facing growing workloads and the need to produce results faster than ever before. Labs are looking for new ways to optimize lab processes and provide quicker results; improve quality while ensuring regulatory compliance

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Conventional Methods Continues to be main work

•Since the 1960s, numerous ingenious innovations have been introduced and used in clinical microbiology laboratories. Parenthetically, none of them allowed microbiologists to abandon the dogma of the pure culture technique,

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Molecular Biology in Infancy

• Molecular biology techniques promise to revolutionize the diagnosis of infectious disease—to date a promise still in its infancy

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Laboratory Medicine Under threatMicrobiology is No exception

•Great questions to many Microbiologists

How much Time we are spending in the Laboratory

Who are doing the Bench work Are laboratories equipped with minimal

requirements of infrastructure and Biosafety?01-02-2016 Dr.T.V.Rao MD 18

NEED FOR AUTOMATION IN INFECTIOUS DISEASES

•Microbes are ever evolving with man we are on the cusp of a dramatic change that will sweep a wave of automation into clinical microbiology laboratories. Threats faced by ecological changes and ever growing misuse of antibiotics for trivial conditions,

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Problems With Traditional Methods• Problems With Traditional Methods

• Cultivation-based methods insensitive for detecting some organisms.

• Cultivation-based methods limited to pathogens with known growth requirements.

• Poor discrimination between microbes with common behavioural features.

• Failure to detect infections caused by uncultivated (e.g., novel) organisms, or organisms that fail to elicit a detectable host immune response.

• Visual appearance of microorganisms is nonspecific.

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Problems With Traditional Methods

• Examples of Failures With Traditional Approaches

• Detection and speciation of slow-growing organisms takes weeks

• (e.g., M. tuberculosis).

• A number of visible microorganisms cannot be cultivated (e.g., Whipple bacillus).

• Diseases presumed to be infectious remain ill-defined with not detected microorganism (e.g., abrupt fever after tick bite).

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NEED FOR MODERN METHODS AUTOMATION

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Genotypic methods• The initiation of new molecular technologies in genomics and proteomics is

shifting traditional techniques for bacterial classification, identification, andcharacterization in the 21st century toward methods based on the elucidation ofspecific gene sequences or molecular components of a cell.

• Genotypic methods of microbe identification include the use of :Nucleic acid probesPCR (RT-PCR, RAPD-PCR)Nucleic acid sequence analysis16s rRNA analysisRFLPPlasmid fingerprinting.

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Advantages of AutomationKnown Advantages

• Maximize resources and automation of the lab to improve efficiency

• Greatly reduce or eliminate errors, and re-work of redundant processes

• Train key personnel and staff on LEAN principles and establish a core knowledge of best practices

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SEPTIC SHOCK A PRIORITY

• Let us think of septic shock as a top priority, at least we receive 10% of the samples, we process are Blood cultures and the number of specimens increase with the expansion of ICU and MICU care, According to the literature, the risk of death from septic shock increases by over 7% with every hour that passes from the onset of shock until the start of targeted therapy. tools in clinical microbiology are primarily based on techniques that evolved 30 to 40 years ago

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RAPID IDENTIFICATION SAVES LIVES

•A number of other studies confirm the urgency of rapid identification of pathogens and its benefit to survival and costs and the effective targeting of antimicrobial therapy, 01-02-2016 Dr.T.V.Rao MD 26

Clinicians start Empirical treatment

• Physicians are forced to deduce the presence of BSI based on clinical symptoms, which are often nonspecific. Subsequently, antibiotic therapy is initiated based on clinical and epidemiologic profiles rather than on

laboratory evidence.

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BacT/ALERT® Culture Media -

•BacT/ALERT® Culture media offers an ideal environment for recovering an array of microorganisms, including bacteria, fungi, and mycobacteria. -

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Reliable Microorganism Recovery

• BacT/ALERT® Culture Media provides a wide range of media bottles to rapidly and reliably recover an array of microorganisms, including bacteria, mycobacteria, fungi, and yeasts from various sample types. BacT/ALERT® Culture Media is FDA-cleared for blood, sterile body fluids, and platelets, and can detect 98% of isolates within 72 hours.* -

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WHO IS ACCOUNTABLE TO RUNNING THE SYSTEM

• 1The physicians should give many clinical details why they wish to culture blood, as automation is fraught with many errors if the coordination fails between treating doctor and laboratory personal the errors with automation are more expensive dangerous with loss of valuable time and the purpose of automation is lost

• 2 All the requests should be identified with caring Doctor or nurse responsible for treatment decision and better with active Mobile contact number for faster communication in case of any growth as the machine alerts every 10 minutes a valuable time is saved for faster decisions

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WHO IS ACCOUNTABLE TO RUNNING THE SYSTEM

•3 The Medical Microbiologists should be well know ledged to identify the contamination and source to make the system running with scientific efficacy

• 4 With experience I wish to say automation needs more knowledge of Medical Microbiologists or the matters will be counterproductive in false identification of contaminants 01-02-2016 Dr.T.V.Rao MD 31

WHO IS ACCOUNTABLE TO RUNNING THE SYSTEM

• 5 A regular audit of the functioning and reporting both by internal and external experts.

• 6 I wish who wish to run the Automation must read from more at Bailey and Scot diagnostic microbiology available in our libraries

• 7 Ill qualified and careless Microbiologists, and technicians are more dangerous as the errors at many stages multiplies faster than traditional methods of culturing leasing greater misuse of Antibiotics

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(MALDI-TOF MS) • Spectrometry (MALDI-TOF MS) is a novel method for the direct

identification of pathogens in blood culture broths, with results available within 2 hours. Although it does not provide antimicrobial susceptibility data identifying the etiologic pathogen, followed by antimicrobial susceptibility testing, is critical in the management of BSIs, as delays in effective antimicrobial therapy can adversely affect patient outcomes. MALDI-TOF MS has significant potential over phenotypic methods, as it is able to detect bacterial pathogens directly from blood culture broths reliably and quickly

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VITEK® MS• VITEK® MS Mass spectrometry microbial identification

system An automated mass spectrometry microbial identification system that uses Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) technology and a comprehensive database of clinically relevant species for results in minutes Robust & accurate ID with Advanced Spectra Classifier Seamless integration of ID/AST results for optimized workflow Complete traceability & flexibility -

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Rapid & clear identification

•VITEK® MS is an innovative, automated microbial identification system that uses MALDI-TOF (Matrix Assisted Laser Desorption Ionization Time-of-Flight) technology. In just minutes, this mass spectrometry technology can provide clear identification at the species, genus and family level. You can provide clinicians with quick information to start appropriate treatment, contributing to overall patient care and outcomes.

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Rapid & clear identification• In just minutes, this mass

spectrometry technology can provide clear identification at the species, genus and family level. You can provide clinicians with quick information to start appropriate treatment, contributing to overall patient care and outcomes –

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The Enigma of Tuberculosis

•Nearly a third of the world's population is estimated to be infected with Mycobacterium tuberculosis. This enormous pool of latently infected individuals poses a major hurdle for global tuberculosis (TB) control. Currently, diagnosis of latent TB infection (LTBI) relies on the tuberculin skin test (TST), a century-old test with known limitations. Smear examination for Acid fast bacilli continues to be Gold standard with many limitations

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Limitations of RNTCP Program

• However the word TB Continues to be least explored disease the treatment in RNTCP as only National programme to control tuberculosis to be challenged by many clinicians who take care of patients however on many fronts the physicians continue to have their own choices and on many fronts empirical treatment continues and the researcher continue to explore MDR tuberculosis as a threat to the many in the society. Research on new TB diagnostic tools has been accelerated over the last few years and the diagnostic pipeline has been growing rapidly as a result

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TB Diagnostic Tools •Research on new TB

diagnostic tools has been accelerated over the last few years and the diagnostic pipeline has been growing rapidly as a result

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Xpert MTB/RIF •Xpert MTB/RIF is an automated, cartridge-based

nucleic amplification assay for the simultaneous detection of TB and rifampicin resistance directly from sputum in under two hours. The technology is based on the GeneXpert platform and was developed as a partnership between the Foundation for Innovative New Diagnostics (FIND), The test simultaneously detects TB and rifampicin drug resistance (a reliable indicator for MDR TB) in sputum.

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My Dear Young Microbiologists

• Most people want to do excellent work. Apathy becomes a problem when team members feel there is no solution or they have no voice

• I wish you are the future leaders in Microbiology for next 40 years hope you all wish for change to live in comfort,

• Assess your role

• Purpose of the speciality

• Try Impress others with hard work and sincerity

• If Medical People do not wish to work some body take you place ?

•NEXT WHAT ?

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Emerging Pathogens: Challenges and Successes of Molecular Diagnostics

•More than 50 emerging and remerging pathogens have been identified during the last 40 years. The feasibility of applying molecular diagnostics to dangerous, fastidious, and uncultivated agents for which conventional tests do not yield timely diagnoses has achieved proof of concept for many agents, but widespread use of cost-effective, validated commercial assays has yet to occur

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Molecular Assays Newer Emerging Trends in Microbiology

• Molecular assays have become widely available for diagnostic microbiology, spurred by technological developments and commercial profit motives. But questions arise when new applications for molecular testing are being introduced. Can these tests replace traditional methods?

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Are we Willing for Change •Can our current

laboratory professionals be trained to perform some of these highly technically complex assays? Will physicians accept the results and change practice appropriately?

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Cost Versus Benefit

•Are the decreased turnaround time and improved sensitivity worth the additional cost?

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Beginning of Polymerase chain reaction

•Newer technologies have transformed the field of microbial diagnosis. Among the first emerging epidemic diseases to be identified by one such method was the hantavirus pulmonary syndrome, a centuries old disease caused by an unknown phlebovirus (Sin Nombre) that was discovered unexpectedly in1993 by the application of a then-novel molecular genetic technique, polymerase chain reaction(PCR).

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MOLECULAR METHODS IN DIAGNOSIS

• The introduction of molecular methods will not only depend on their performance for each individual microorganism, but also on the clinical relevance of the diagnostic question asked, the prevalence of the clinical problem and whether the new methods are added to the procedures in use or will replace them. Therefore no general rules can be proposed, strategies have to be elaborated for each infectious agent or clinical syndrome.

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WHEN WE REALLY NEED MOLECULAR METHODS ?

•Molecular diagnosis is most appropriate for infectious agents that are difficult to detect, identify, or test for susceptibility in a timely fashion with conventional methods.

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THERE IS AN URGENT NEED FOR MOLECULAR METHODS IN

• Strategies concerning the use of molecular diagnostic techniques for the diagnosis of Mycobacterium tuberculosis, Chlamydia trachomatis, meningo-encephalitis syndrome and respiratory infection

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Nucleic acid probes• Nucleic acid hybridization is one of the most powerful tools available for

microbe identification.

• Hybridization detects for a specific DNA sequence associated with anorganism.

• The process uses a nucleic acid probe which is specific for that particularorganism.

• The target DNA (from the organism) is attached to a solid matrix such as anylon or nitrocellulose membrane.

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Nucleic Acid Probes• A single stranded probe is added and if there

is sequence complementality between thetarget and the probe a positive hybridizationsignal will be detected.

• Hybridization is detected by areporter molecule (radioactive,fluorescent, chemiluminescent)which is attached to the probe.

• Nucleic acid probes have beenmarketed for the identification ofmany pathogens such as N.gonorrhoeae.

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Advantages of Nucleic Acid Probes•Nucleic acid probes has many advantages over

immunological methods.

•Nucleic acid are more stable at high temperature,pH, and in the presence of organic solvents and otherchemicals.

• This means that the specimen can be treated veryharshly to destroy interfering materials.

•Nucleic acid probes can be used to identifymicroorganisms which are no longer alive.

• Furthermore nucleic acid probes are more specificthan antibodies.01-02-2016 Dr.T.V.Rao MD

DIAGNOSTIC MICROBIOLOGY CHANGING FROM PHENOTYPIC METHODS TO MOLECULAR METHODS

• In hospital epidemiology, the use of such techniques has already provided tests with exceptional discriminatory power. Molecular techniques allow more efficient typing of all pathogens, and permit discrimination between strains of organisms that were previously phenotypically identical or uncharacterizable. Currently, cost and complexity limit the applicability of these techniques; however, they are likely to be developed for routine laboratory use in the next decade, and their impact will be considerable.

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EMERGING MOLECULAR METHODS IN DIAGNOSIS

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NEW TECHNOLOGIES – REAL TIME ASSAYS

• The Real Time assays are proving to better technologies

• 1 Rapid

• 2 Quantitative measurement

• 3 Lower contamination rate

• 4 Higher sensitivity

• 5 Higher specificity

• 6 Easy standardization

• Now a new gold standard for rapid diagnosis of virus infection in the acute phase samples.

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WE HAVE NO POLICY TO IMPROVE THE DIAGNOSTICS IN INDIA?

•We in the country do not have any fixed guidelines or acts how laboratories can improve unlike some of the developed countries While ensuring accurate data is the critical first step to technology implementation. Faster time-to-results and the detection of disease at an earlier stage enable patients to receive the appropriate treatment more quickly. A closer link is being forged between diagnostic tests and treatment

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Who can afford it? • Fluorescence microscopes, thermocyclers, qPCR

machines, hybridisation ovens, automated expert systems, specialised reagents - these are the more expensive requirements of the modern microbiology laboratory. In some regions of the world uptake of the new technologies has been slow. For resource-poor areas, the obstacles can seem insurmountable because significant funding must be allocated for upgrading laboratory infrastructure and training of staff as well as major equipment purchases

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Long way to go in Our approach

• Because multidrug-resistant gram-negative rods are among the most important causes of healthcare-acquired infections today and because well-studied or commercial molecular methods are not yet available for their detection

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OUR VISION TO FUTURE DIAGNOSIS OF INFECTIOUS DISEASES

•With the ability to test for an unlimited number of potential pathogens simultaneously, next-generation sequencing has the potential to revolutionize infectious diseases diagnostics

•In the microbiology laboratory, this technology will likely replace the traditional “one test, one bug” approach to pathogen diagnostics

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WHY WE MUST BE FAMILIAR WITH MOLECULAR METHODS

• In Many Developed countries several Diagnostic methods are switched on to Molecular Methods.

• No scientific journal is willing to accept or publish any article without incorporation of Molecular Methods.

• Antibiotic drug resistance is a growing concern, to the world, unless molecular identification is performed on responsible genetic mechanisms no effective scientific conclusions can be drawn to contain the spread.

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Are we ignoring Anaerobes ?

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Tele Diagnosis

CDC helps in Digital diagnosis ➲The CDC now offers tele diagnosis to help laboratories diagnose

malaria and other parasitic diseases. When laboratories are not certain about identifying parasites on a slide, they can e-mail to the CDC images of the suspected parasites. Experts then review the images and discuss findings with the submitting lab within only a few hours, allowing near real-time diagnosis as well as an opportunity for training in microscopic diagnosis.

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A long parasite of 15 cm was extracted without damage to the physical structure

•An approximately 15 cm long filamentous macroscopic parasite was extracted sent intact in Normal saline, to Microbiology

Histopathology sections and Photomicrograph

As reported from CDC – Atlanta USA

• Based on the images, we agree this is a female Dirofilaria (possibly D. repens in India), as indicated by tall, polymyarian musculature, external cuticular ridges, and paired reproductive tubes.

Documentation in Microbiology

WHONET is a Windows-based data WHONET is a Windows-based database software developed for the management of microbiology laboratory data and the analysis of antimicrobial susceptibility test results.

•The software developed for the management of microbiology laboratory data and the analysis of antimicrobial susceptibility test results.

Data can be exchanged globally

• To enhance local use of laboratory data for guiding therapy, assisting infection control, characterizing resistance epidemiology and identifying laboratory testing errors; to promote collaboration in surveillance activities through the exchange of data.

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Connects to the World through WWW

• These tools enable any microbiology laboratory to put its test results into a database and conduct analyses to support local infection control and antibiotic use. Laboratories can also upload files created by WHONET, to feed into national or other multi-centre surveillance networks and to inform drug policy. Such surveillance programs are now in place in many countries.

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Laboratory Automation Advanced Beyond imagination

• Laboratory automation has already made dramatic changes to the way in which researchers approach their work. It will clearly provide new tools for increasing productivity in the future What is ultimately possible seems to be limited only by the creative genius of researchers working in the laboratory to define their needs and companies focusing on providing creative and user-friendly solutions to those problems and challenges.

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Robotics Entering Diagnostic Laboratories

• Advances to come include programmable, automated work stations that can perform a multitude of tasks, extremely sophisticated robotics that can perform tasks once restricted to humans, and artificial intelligence systems on chips that can learn from their experiences much like ordinary humans.

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Decreasing human Role

• Some laboratories have even enlisted the services of fully functional robotic systems to replace jobs once done manually during an eight-hour shift into procedures that require very little human intervention and operate (at least in theory) 24 hours a day, 7 days a week, 365 days a year. "

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World First for Malaria - mobile phone diagnosis now available

• xRapid is a world first in mobile health, providing automatic diagnosis of malaria via an iPhone app. It is the first commercially available mobile app that has the functionality to quickly and accurately diagnose a major disease.

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ARE WE READY FOR CHANGE TO AUTOMATION ?

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THE FUTURE OF DIAGNOSTIC MICROBIOLOGY IS CHANGING

• The physical structure of laboratories, staffing patterns, work flow, and turnaround time have all been profoundly influenced by technical advances. The implementation of nucleic acid amplification-based molecular techniques provides complementary, rapid, and on-demand diagnosis services. These changes will continue, and lead diagnostic microbiology inevitably to a modern discipline, which can face many challenges in the future.

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Interesting quote on

AUTOMATIONDonald A. Norman University of California, San Diego

• The automation is at an intermediate level of intelligence, powerful enough to take over control that used to be done by people, but not powerful enough to handle all abnormalities. Moreover, its level of intelligence is insufficient to provide the continual, appropriate feedback that occurs naturally among human operators. This is the source of the current difficulties. To solve this problem, the automation should either be made less intelligent or more so, but the current level is quite inappropriate.01-02-2016 Dr.T.V.Rao MD 76

How we can improve our Diagnostic Microbiology

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THANK YOU ALL WE WILL PROGRESS ONE DAY ?

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