CHALLENGES IN DIAGNOSTIC MICROBIOLOGY

(Overcoming with Newer Approaches in Diagnostic Microbiology)

Dr.T.V.Rao MD
When we are Young in the Microbiology departments the matters were
simple with many traditional approaches in diagnosis of Bacterial, Parasitic and Fungal infections,
solving the problems, Today the Diagnostic Microbiology is ignited with onset of AIDS, as many
common, uncommon and rare Microbes starting engulfing the humans, Profession started to look
ahead to the future. 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. This pivotal role is made possible by the adoption of rational sampling, point-of-care
tests, extended automation and other new technologies, including mass spectrometry for colony
identification, real-time genomics for isolate characterization, and versatile and permissive culture
systems. However many of our diagnostic laboratories are in primitive stage, However we have a
larger responsibility to improve the Human care with optimal Diagnostic Microbiology

INFECTIONS AND ENVIRONMENT - The emergence of new infections and spread dependent on
migration of Humans to Urban in search of employment and livelihood, It would seem that any
urbanisation drives up the emergence of infectious diseases in those areas, Climate change affects
infectious diseases directly and indirectly. Meteorological factors including temperature,
precipitation, humidity and radiation influence infectious disease by modulating pathogen, host and
transmission pathways. Meteorological disasters such as droughts and floods directly impact the
outbreak and transmission of infectious diseases. Climate change indirectly impacts infectious
diseases by altering the ecological system, including its underlying surface and vegetation
distribution.

EMERGING CHALLENGES TO MEDICAL MICROBIOLOGISTS

What the Hospitals and Administrators expect from us - Todays 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; However 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 techniques. Never forget Laboratory Medicine Under
threat Microbiology is no exception- Great questions to many Microbiologists.

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
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).

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

NEED FOR GENOMIC 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 probes
PCR (RT-PCR, RAPD-PCR)
Nucleic acid sequence analysis
16s rRNA analysis
RFLP

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

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.

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
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 Gene pert platform and was developed as a partnership between the
Foundations for Innovative New Diagnostics (FIND), the test simultaneously detects TB and
rifampicin drug resistance (a reliable indicator for MDR TB) in sputum.

Emerging Pathogens: Challenges and Success of Molecular Diagnostics - More than 50

emerging and re-emerging 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 Molecular Assays.
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.

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.

METHODS WE SHOULD LOOK TO IMPROVE THE DIAGNOSTIC MICROBIOLOGY

1 BacT/ALERT Culture Media
2 VITEK MS
3 MALDI-TOF MS)
REAL TIME PCR
Real-time PCR has established itself as a sensitive and specific qualitative and quantitative technique
that has become important to all areas of microbiology. The use of real-time PCR in microbial
diagnostics has increased to the point where it has evolved from a novelty into a mature and
essential technology for the field. In doing so, real-time PCR has driven significant changes in the way
we detect microbes. Real-time PCR has engendered wider acceptance of the PCR technique due to
its improved rapidity, sensitivity, reproducibility and the considerably reduced risk of carry-over
contamination.

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.

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.

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.
TIME TO MOVE FROM PRACTICE TO GOOD PRACTICES
We are moving from just doing the basic work in Diagnostic Microbiology to opting for Best
practices, as every division of Medicine moving from just attending and treating the patients to
practice best practices, It is a new trend in Medicine, However the Practicing Microbiologists should
STRIVE TO IMPROVE DIAGNOSTIC MICROBIOLOGY for BEST PRACTICES. Planning is an essential step
in any exercise to rank the risk of communicable disease threats. Establishing the objectives of the
exercise enables the selection of an appropriate methodology that is fit for purpose

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. Furthermore, it enables others to replicate,
develop and improve upon previous practice, leading to improvements in methodologies
Ref 1Emerging infectious diseases and cities, By James Byrne Scientific American
Ref 2Emerging challenges in Infectious Diseases CDC resources
Dr.T.V.Rao MD Professor of Microbiology
Email doctortvrao@gmail.com

The Document made for improving our vision to Diagnostic Microbiology for the
changing needs in Infectious Diseases
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