01/05/2023 Dr.T.V.Rao MD 1

EMERGING CHALLENGES IN DIAGNOSTIC MICROBIOLOGY

(Overcoming with Newer Approaches in Diagnostic Microbiology)Dr.T.V.Rao MD

01/05/2023 Dr.T.V.Rao MD 2

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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The Great Challenge to Medical Microbiologists

•The critical role of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team.

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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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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.. 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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It is not Just Reporting but challenged with

Interpretation•Unlike other areas of the diagnostic laboratory, clinical microbiology is a science of interpretive judgment that is becoming more complex, not less

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Clinical Relevance is Back Bone of Microbiology

•Physicians need confidence that the results provided by the microbiology laboratory are accurate, significant, and clinically relevant. Anything less is below the community standard of care

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Entry of New reasons for concern in Infectious diseases • But the very nature of

clinical microbiology has undergone profound changes in the past few decades. Hitherto unknown microorganisms and viruses have appeared, often as the result of human activities with environmental impacts

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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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Current Challenges in Medical Microbiology

• The task of identifying, isolating, and evaluating therapies for these emerging host-harmful entities has become part and parcel of the present function of clinical microbiologists, aided frequently by the tools of molecular biology.

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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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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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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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EMERGING INFECTIOUS DISEASESthey are overtaking us

Microbes and vectors swim in the evolutionary stream, and they swim faster than we do. Bacteria reproduce every 30 minutes. For them, a millennium is compressed into a fortnight. They are fleet afoot, and the pace of our research must keep up with them, or they will overtake us. Microbes were here on earth 2 billion years before humans arrived, learning every trick for survival, and it is likely that they will be here 2 billion years after we department. (Krause 1998).

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Climate change Promotes Emerging Infections

•Climate change is increasingly becoming a concern as a factor in the emergence of infectious diseases. As Earth's climate warms and habitats are altered, diseases can spread into new geographic areas.

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• The changing landscape of infectious diseases over the past 2 decades has astounded the medical PROFESSIONALS and the PUBLIC. Emerging pathogens that cause new diseases (e.g., severe acute respiratory syndrome–associated coronavirus [SARS-CoV]), newly recognized microbial agents of known diseases (e.g., human metapneumovirus), and rapidly evolving pathogens (e.g., influenza viruses) all contribute to this seismic shift

Infectious diseases are not Static but New are Emerging

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Changes in human behavior, food habits and

environment•Unsanitary food

preparation, exposure of humans to disease vectors and reservoirs, and ecological changes that alter the composition and size of insect vectors and animal reservoirs.

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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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Role of Microbiology Crucial link

• The importance of Microbiology laboratories rests with prompt identification and reporting can cure with simple remedies, as most of the emerging infections respond to several drugs and choice lies with optimal selection based on Antibiograms, as resistance is not a menace with emerging, newer and uncommon isolates

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

traditional techniques for bacterial classification, identification, and characterization in the 21st century toward methods based on the elucidation of specific 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 Automation

Known 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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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,

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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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(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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We Can start using ABIS on line resources

•The program allows a great flexibility in choosing biochemical tests and is an alternative to commercial systems, code-books or identification tables.•The software is linked with a germs Encyclopaedia, the Kauffman-White scheme for Salmonella serovars identification, Lancefield grouping for streptococci and an antibiogram interpreter.

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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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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.

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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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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,

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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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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, meningitis encephalitis syndrome and respiratory infection

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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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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

01/05/2023 Dr.T.V.Rao MD

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 an organism.

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

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

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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 other chemicals.• This means that the specimen can be treated very

harshly to destroy interfering materials.•Nucleic acid probes can be used to identify

microorganisms which are no longer alive.• Furthermore nucleic acid probes are more specific

than antibodies.

Documentation in Microbiology 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.

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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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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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Tele Diagnosis in Parasitology

• 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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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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NEVER FORGET LABORATORY REPORTING IS THE MIRROR

•NEVER FORGET LABORATORY REPORTING IS THE MIRROR OF strengths and weaknesses - Reporting results means most of the process can be understood and assessed by multiple stakeholders particularly our physicians and surgeons.

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ARE WE READY FOR CHANGE TO

AUTOMATION ?

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Interesting quote on AUTOMATION

Donald 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.

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We Need Quality Control at Many stages in the day to day working in our Laboratories

•A high priority for future global consortiums of epidemiology and biomedical research centres will be to initiate or build upon existing systems in sentinel hospitals in developing countries for the international monitoring and reporting of antimicrobial susceptibility data. Two systems that offer a foundation of international linkages are CDC’s International Nosocomial Surveillance, Program for Emerging Antimicrobial Resistance and the WHO Antimicrobial Resistance Monitoring Program •

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We Need Monitoring in all our diagnostic work

•Monitoring Program. The international and national objectives of these programs depend on conducting proper, quality-controlled, antimicrobial susceptibility testing and promoting the use of resistance data to guide antimicrobial therapy

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There are sea of changes in Microbiology

Are we in the Right Direction

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References • Evidence based diagnostic microbiology: has its time come.

V Sintchenko and G L Gilbert ; J Clin Pathol 2011• Emerging infectious diseases and cities, By James Byrne

Scientific American • Clinical Microbiology in Developing Countries, Lennox K.

Archibald* and L. Barth Reller† *Centers for Disease Control and Prevention, Atlanta, Georgia, USA, and †Duke University Medical Center, Durham, North Carolina, USA

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