Lecture 1

Clinical Microbiology
Laboratory
Trends and Prospects
 Modern clinical MB
laboratory design
 Increase our capabilities in
microorganism detection and
identification!
 Current trends
 Point of care devices or rapid
diagnostic tests (RDTs): ex.,
rapid PCR (rPCR), hybridization
in situ, MALDI-TOF mass
spectrometry, serological tests
for Ag/Ab detection
 Total laboratory automation
(→ VITEC, BACTEC, WASP)
Monitoring blood culture system
 Point-of-care testing (POCT), or
bedside testing is defined as medical
diagnostic testing at or near the point of
care—that is, at the time and place of
patient care.
 This contrasts with the historical pattern
in which testing was wholly or mostly
confined to the medical laboratory, which
entailed sending off specimens away from
the point of care and then waiting hours
or days to learn the results.
 RDTs example of use
 Coagulase-negative staphylococci
(CoNS) are part of the normal skin flora
and often represent
specimen contamination when isolated
from a blood culture. This is
particularly common when two sets of
blood cultures were taken from
different sites and only 1 of the 2 is
positive.
 The problem is that in the absence of RDTs,
clinicians typically have to cover patients
empirically with antibiotics for several days
until traditional microbiology testing
methods produce organism identification.

 Using RDTs, CoNS in blood cultures can be

identified much earlier on and unnecessary
exposure to potentially toxic antibiotics can
be avoided.
 Itis recommended to use of an
RDT called peptide nucleic acid
fluorescence in situ
hybridization (PNA FISH) for
rapid identification of
Staphylococcus aureus versus
CoNS from blood cultures
positive with Gram-positive
cocci in clusters.
 Principal paradigm shift
 Growth of genomic approaches
- PCR, genome sequencing
 Use of proteomic approach -
matrix‐assisted laser
desorption ionization (MALDI)
combined with time of flight
(ToF) mass spectrometry (MS)
 Currently available methods
 1. Conventional biochemical
systems (API-systems, Motley
Hiss row, DDM, etc.)
 2. Molecular identification
methods (=molecular-genetic)
 3. Fatty acid analysis with gas

chromatography
 4. Protein mass spectrometry
 5. Automated systems for the
phenotypic identification
 6. Serological/immunological
methods
Conventional identification methods
 rely on phenotypic
characteristics
 largely based on biochemical
reactions such as carbohydrate
utilization and enzymatic
modification of substrates
 commercially available in
manual or automated formats
ENDO AGAR
 Automated systems for the phenotypic
identification of aerobic bacteria
►Use miniaturized biochemical panels
that are incubated in the instrument and
read at specified intervals. Software
programs interpret the data collected
from the results of the phenotypic
reactions and generate a biochemical
profile. The profiles are compared to
databases to identify of the
microorganism
Automated systems examples
 Vitek 2 (bioMérieux)
 MicroScan (Siemens Healthcare
Diagnostics)
 Phoenix (BD Diagnostic Systems)
 The MicroScan WalkAway is an
automated bacterial identification and
susceptibility testing system
VITEC, VITEC-2 analyser
 The system successively measured
colorimetric or turbidometric changes
within individual card wells to determine
positive and negative endpoints
VITEC Card well
VITEC
 Automated and semiautomated
technology in microbiology which has
seen great advances in recent years.
Molecular identification methods
 direct probe hybridization
 various iterations of nucleic acid
amplification and detection
 DNA microarrays

 DNA sequencing
 MALDI‐ToF/MS
 emerging technology for
identification that is rapid,
accurate, and inexpensive
 based on mass spectrometry

 can be used to classify and

identify bacteria
MALDI-TOF mass spectrometry
 Currently microorganisms are best
identified using 16S rRNA and 18S
rRNA gene sequencing. However,
in recent years matrix assisted
laser desorption ionization-time of
flight mass spectrometry (MALDI-
TOF MS) has emerged as a
potential tool for microbial
identification and diagnosis
 The technology has been readily used by
microbiologists who have reported usage
of MALDI-TOF MS for a number of
purposes like, microbial identification and
strain typing, epidemiological studies,
detection of biological warfare agents,
detection of water- and food-borne
pathogens, detection of antibiotic
resistance and detection of blood and
urinary tract pathogens etc.
 The limitation of the technology is
that identification of new isolates
is possible only if the spectral
database contains peptide mass
fingerprints of the type strains of
specific
genera/species/subspecies/strains