Molecular Approaches to Diagnosing and Managing Infectious Diseases:
Practicality and Costs
Michael A. Pfaller University of Iowa College of Medicine, Iowa City,
Iowa, USA
[Emerging Infectious Diseases (7)2, 2001. Centers for Disease Control]
Abstract
As molecular techniques for identifying and detecting microorganisms
in the clinical microbiology laboratory have become routine,
questions about the cost of these techniques and their contribution
to patient care need to be addressed. 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.
Introduction
The tools of molecular biology have proven readily adaptable for use
in the clinical diagnostic laboratory and promise to be extremely
useful in diagnosis, therapy, and epidemiologic investigations and
infection control[1,2]. Although technical issues such as ease of
performance, reproducibility, sensitivity, and specificity of
molecular tests are important, cost and potential contribution to
patient care are also of concern[3]. Molecular methods may be an
improvement over conventional microbiologic testing in many ways.
Currently, their most practical and useful application is in
detecting and identifying infectious agents for which routine growth-
based culture and microscopy methods may not be adequate[4-7].
Nucleic acid-based tests used in diagnosing infectious diseases use
standard methods for isolating nucleic acids from organisms and
clinical material and restriction endonuclease enzymes, gel
electrophoresis, and nucleic acid hybridization techniques to analyze
DNA or RNA[6]. Because the target DNA or RNA may be present in very
small amounts in clinical specimens, various signal amplification and
target amplification techniques have been used to detect infectious
agents in clinical diagnostic laboratories[5,6]. Although mainly a
research tool, nucleic acid sequence analysis coupled with target
amplification is clinically useful and helps detect and identify
previously uncultivatable organisms and characterize antimicrobial
resistance gene mutations, thus aiding both diagnosis and treatment
of infectious diseases[5,8,9]. Automation and high-density
oligonucleotide probe arrays (DNA chips) also hold great promise for
characterizing microbial pathogens[6].
Although most clinicians and microbiologists enthusiastically welcome
the new molecular tests for diagnosing infectious disease, the high
cost of these tests is of concern[3]. Despite the probability that
improved patient outcome and reduced cost of antimicrobial agents and
length of hospital stay will outweigh the increased laboratory costs
incurred through the use of molecular testing, such savings are
difficult to document[3,10,11]. Much of the justification for
expenditures on molecular testing is speculative[11]; however, the
cost of equipment, reagents, and trained personnel is real and
substantial, and reimbursement issues are problematic[3,11]. Given
these concerns, a facility's need for molecular diagnostic testing
for infectious diseases should be examined critically by the affected
clinical and laboratory services. In many instances, careful
overseeing of test ordering and prudent use of a reference laboratory
may be the most viable options.
Practical Applications of Molecular Methods in the Clinical
Microbiology Laboratory
Commercial kits for the molecular detection and identification of
infectious pathogens have provided a degree of standardization and
ease of use that has facilitated the introduction of molecular
diagnostics into the clinical microbiology laboratory (Table 1). The
use of nucleic acid probes for identifying cultured organisms and for
direct detection of organisms in clinical material was the first
exposure that most laboratories had to commercially available
molecular tests. Although these probe tests are still widely used,
amplification-based methods are increasingly employed for diagnosis,
identification and quantitation of pathogens, and characterization of
antimicrobial-drug resistance genes. Commercial amplification kits
are available for some pathogens (Table 1), but some clinically
important pathogens require investigator-designed or "home-brew"
methods (Table 2). In addition, molecular strain typing, or
genotyping, has proven useful in guiding therapeutic decisions for
certain viral pathogens and for epidemiologic investigation and
infection control[2,12].
Detection and Identification of Pathogens Without Target Amplification
Commercial kits containing non-isotopically labeled nucleic acid
probes are available for direct detection of pathogens in clinical
material and identification of organisms after isolation in culture
(Table 1). Use of solution-phase hybridization has allowed tests to
be performed singly or in batches in a familiar microwell format.
Although direct detection of organisms in clinical specimens by
nucleic acid probes is rapid and simple, it suffers from lack of
sensitivity. Most direct probe detection assays require at least 104
copies of nucleic acid per microliter for reliable detection, a
requirement rarely met in clinical samples without some form of
amplification. Amplification of the detection signal after probe
hybridization improves sensitivity to as low as 500 gene copies per
microliter and provides quantitative capabilities. This approach has
been used extensively for quantitative assays of viral load (HIV,
hepatitis B virus [HBV] and hepatitis C virus [HCV]) (Table 1) but
does not match the analytical sensitivity of target amplification-
based methods, such as polymerase chain reaction (PCR), for detecting
organisms.
The commercial probe systems that use solution-phase hybridization
and chemiluminescence for direct detection of infectious agents in
clinical material include the PACE2 products of Gen-Probe and the
hybrid capture assay systems of Digene and Murex (Table 1). These
systems are user friendly, have a long shelf life, and are adaptable
to small or large numbers of specimens. The PACE2 products are
designed for direct detection of both Neisseria gonorrhoeae and
Chlamydia trachomatis in a single specimen (one specimen, two
separate probes). The hybrid capture systems detect human papilloma
virus (HPV) in cervical scrapings, herpes simplex virus (HSV) in
vesicle material, and cytomegalovirus (CMV) in blood and other
fluids. All these tests have demonstrated sensitivity exceeding that
of culture or immunologic methods for detecting the respective
pathogens but are less sensitive than PCR or other target
amplification-based methods.
The signal amplification-based probe methods for detection and
quantitation of viruses (HBV, HCV, HIV) are presented in an enzyme
immunoassay-like format and include branched chain DNA probes
(Chiron) and QB replicase (Gene-Trak) methods (Table 1). These
methods are not as sensitive as target amplification-based methods
for detection of viruses; however, the quantitative results have
proven useful for determining viral load and prognosis and for
monitoring response to therapy[13].
Probe hybridization is useful for identifying slow-growing organisms
after isolation in culture using either liquid or solid media.
Identification of mycobacteria and other slow-growing organisms such
as the dimorphic fungi (Histoplasma capsulatum, Coccidioides immitis,
and Blastomyces dermatitidis) has certainly been facilitated by
commercially available probes. All commercial probes for identifying
organisms are produced by Gen-Probe and use acridinium ester-labeled
probes directed at species-specific rRNA sequences (Table 1). Gen-
Probe products are available for the culture identification of
Mycobacterium tuberculosis, M. avium-intracellulare complex, M.
gordonae, M. kansasii, Cryptococcus neoformans, the dimorphic fungi
(listed above), N. gonorrhoeae, Staphylococcus aureus, Streptococcus
pneumoniae, Escherichia coli, Haemophilus influenzae, Enterococcus
spp., S. agalactiae, and Listeria monocytogenes. The sensitivity and
specificity of these probes are excellent, and they provide species
identification within one working day. Because most of the bacteria
listed, plus C. neoformans, can be easily and efficiently identified
by conventional methods within 1 to 2 days, many of these probes have
not been widely used. The mycobacterial probes, on the other hand,
are accepted as mainstays for the identification of M. tuberculosis
and related species[7].
Nucleic Acid Amplification
Nucleic acid amplification provides the ability to selectively
amplify specific targets present in low concentrations to detectable
levels; thus, amplification-based methods offer superior performance,
in terms of sensitivity, over the direct (non-amplified) probe-based
tests. PCR (Roche Molecular Systems, Branchburg, NJ) was the first
such technique to be developed and because of its flexibility and
ease of performance remains the most widely used molecular diagnostic
technique in both research and clinical laboratories. Several
different amplification-based strategies have been developed and are
available commercially (Table 1). Commercial amplification-based
molecular diagnostic systems for infectious diseases have focused
largely on systems for detecting N. gonorrhoeae, C. trachomatis, M.
tuberculosis, and specific viral infections (HBV, HCV, HIV, CMV, and
enterovirus) (Table 1). Given the adaptability of PCR, numerous
additional infectious pathogens have been detected by investigator-
developed or home-brew PCR assays[5] (Table 2). In many instances,
such tests provide important and clinically relevant information that
would otherwise be unavailable since commercial interests have been
slow to expand the line of products available to clinical
laboratories. In addition to qualitative detection of viruses,
quantitation of viral load in clinical specimens is now recognized to
be of great importance for the diagnosis, prognosis, and therapeutic
monitoring for HCV, HIV, HBV, and CMV[13]. Both PCR and nucleic acid
strand-based amplification systems are available for quantitation of
one or more viruses (Table 1).
The adaptation of amplification-based test methods to commercially
available kits has served to optimize user acceptability, prevent
contamination, standardize reagents and testing conditions, and make
automation a possibility. It is not clear to what extent the levels
of detection achievable by the different amplification strategies
differ. None of the newer methods provides a level of sensitivity
greater than that of PCR. In choosing a molecular diagnostic system,
one should consider the range of tests available, suitability of the
method to workflow, and cost[6]. Choosing one amplification-based
method that provides testing capabilities for several pathogens is
certainly practical.
Amplification-based methods are also valuable for identifying
cultured and non-cultivatable organisms[5]. Amplification reactions
may be designed to rapidly identify an acid-fast organism as M.
tuberculosis or may amplify a genus-specific or "universal" target,
which then is characterized by using restriction endonuclease
digestion, hybridization with multiple probes, or sequence
determination to provide species or even subspecies delineation
[4,5,14]. Although identification was initially applied to slow-
growing mycobacteria, it has applications for other pathogens that
are difficult or impossible to identify with conventional methods.
Detecting Antimicrobial-Drug Resistance
Molecular methods can rapidly detect antimicrobial-drug resistance in
clinical settings and have substantially contributed to our
understanding of the spread and genetics of resistance[9].
Conventional broth- and agar-based antimicrobial susceptibility
testing methods provide a phenotypic profile of the response of a
given microbe to an array of agents. Although useful for selecting
potentially useful therapeutic agents, conventional methods are slow
and fraught with problems. The most common failing is in the
detection of methicillin resistance in staphylococci, which may be
expressed in a very heterogeneous fashion, making phenotypic
characterization of resistance difficult[9,15]. Currently, molecular
detection of the resistance gene, mec A, is the standard against
which phenotypic methods for detection of methicillin resistance are
judged[9,15,16].
Molecular methods may be used to detect specific antimicrobial-drug
resistance genes (resistance genotyping) in many organisms (Table 3)
[8,9]. Detection of specific point mutations associated with
resistance to antiviral agents is also increasingly important[17,18].
Screening for mutations in an amplified product may be facilitated by
the use of high-density probe arrays (Gene chips)[6].
Despite its many potential advantages, genotyping will not likely
replace phenotypic methods for detecting antimicrobial-drug
resistance in the clinical laboratory in the near future. Molecular
methods for resistance detection may be applied directly to the
clinical specimen, providing simultaneous detection and
identification of the pathogen plus resistance characterization[9].
Likewise, they are useful in detecting resistance in viruses, slow-
growing or nonviable organisms, or organisms with resistance
mechanisms that are not reliably detected by phenotypic methods
[9,19]. However, because of their high specificity, molecular methods
will not detect newly emerging resistance mechanisms and are unlikely
to be useful in detecting resistance genes in species where the gene
has not been observed previously[19]. Furthermore, the presence of a
resistance gene does not mean that the gene will be expressed, and
the absence of a known resistance gene does not exclude the
possibility of resistance from another mechanism. Phenotypic
antimicrobial susceptibility testing methods allow laboratories to
test many organisms and detect newly emerging as well as established
resistance patterns.
Molecular Epidemiology
Laboratory characterization of microbial pathogens as biologically or
genetically related is frequently useful in investigations[12,20,21].
Several different epidemiologic typing methods have been applied in
studies of microbial pathogens (Table 4). The phenotypic methods have
occasionally been useful in describing the epidemiology of infectious
diseases; however, they are too variable, slow, and labor-intensive
to be of much use in most epidemiologic investigations. Newer DNA-
based typing methods have eliminated most of these limitations and
are now the preferred techniques for epidemiologic typing. The most
widely used molecular typing methods include plasmid profiling,
restriction endonuclease analysis of plasmid and genomic DNA,
Southern hybridization analysis using specific DNA probes, and
chromosomal DNA profiling using either pulsed-field gel
electrophoresis (PFGE) or PCR-based methods[12,20]. All these methods
use electric fields to separate DNA fragments, whole chromosomes, or
plasmids into unique patterns or fingerprints that are visualized by
staining with ethidium bromide or by nucleic acid probe hybridization
(Figure). Molecular typing is performed to determine whether
different isolates give the same or different results for one or more
tests. Epidemiologically related isolates share the same DNA profile
or fingerprint, whereas sporadic or epidemiologically unrelated
isolates have distinctly different patterns (Figure). If isolates
from different patients share the same fingerprint, they probably
originated from the same clone and were transmitted from patient to
patient by a common source or mechanism.
Figure. Pulsed-field gel electrophoresis (PFGE) profiles of
Staphylococcus aureus isolates digested with Sma 1. A variety of PFGE
profiles are demonstrated in these 23 isolates.
Molecular typing methods have allowed investigators to study the
relationship between colonizing and infecting isolates in individual
patients, distinguish contaminating from infecting strains, document
nosocomial transmission in hospitalized patients, evaluate
reinfection versus relapse in patients being treated for an
infection, and follow the spread of antimicrobial-drug resistant
strains within and between hospitals over time[12]. Most available
DNA-based typing methods may be used in studying nosocomial
infections when applied in the context of a careful epidemiologic
investigation[12,21]. In contrast, even the most powerful and
sophisticated typing method, if used indiscriminately in the absence
of sound epidemiologic data, may provide conflicting and confusing
information.
Financial Considerations
Molecular testing for infectious diseases includes testing for the
host's predisposition to disease, screening for infected or colonized
persons, diagnosis of clinically important infections, and monitoring
the course of infection or the spread of a specific pathogen in a
given population. It is often assumed that in addition to improved
patient care, major financial benefits may accrue from molecular
testing because the tests reduce the use of less sensitive and
specific tests, unnecessary diagnostic procedures and therapies, and
nosocomial infections[11]. However, the inherent costs of molecular
testing methods, coupled with variable and inadequate reimbursement
by third-party payers and managed-care organizations, have limited
the introduction of these tests into the clinical diagnostic
laboratory.
Not all molecular diagnostic tests are extremely expensive. Direct
costs vary widely, depending on the test's complexity and
sophistication. Inexpensive molecular tests are generally kit based
and use methods that require little instrumentation or technologist
experience. DNA probe methods that detect C. trachomatis or N.
gonorrhoeae are examples of low-cost molecular tests. The more
complex molecular tests, such as resistance genotyping, often have
high labor costs because they require experienced, well-trained
technologists. Although the more sophisticated tests may require
expensive equipment (e.g., DNA sequencer) and reagents, advances in
automation and the production of less-expensive reagents promise to
decrease these costs as well as technician time. Major obstacles to
establishing a molecular diagnostics laboratory that are often not
considered until late in the process are required licenses, existing
and pending patents, test selection, and billing and reimbursement
[22].
Reimbursement issues are a major source of confusion, frustration,
and inconsistency. Reimbursement by third-party payers is confounded
by lack of Food and Drug Administration (FDA) approval and Current
Procedural Terminology (CPT) codes for many molecular tests. In
general, molecular tests for infectious diseases have been more
readily accepted for reimbursement; however, reimbursement is often
on a case-by-case basis and may be slow and cumbersome. FDA approval
of a test improves the likelihood that it will be reimbursed but does
not ensure that the amount reimbursed will equal the cost of
performing the test.
Perhaps more than other laboratory tests, molecular tests may be
negatively affected by fee-for-service managed-care contracts and
across-the-board discounting of laboratory test fees. Such measures
often result in reimbursement that is lower than the cost of
providing the test. Although molecular tests may be considered a
means of promoting patient wellness, the financial benefits of
patient wellness are not easily realized in the short term[11].
Health maintenance organizations (HMOs) and managed-care
organizations often appear to be operating on shorter time frames,
and their administrators may not be interested in the long-term
impact of diagnostic testing strategies.
Molecular screening programs for infectious diseases are developed to
detect symptomatic and asymptomatic disease in individuals and
groups. Persons at high risk, such as immunocompromised patients or
those attending family planning or obstetrical clinics, are screened
for CMV and Chlamydia, respectively. Likewise, all blood donors are
screened for bloodborne pathogens. The financial outcome of such
testing is unknown. The cost must be balanced against the benefits of
earlier diagnosis and treatment and societal issues such as disease
epidemiology and population management.
One of the most highly touted benefits of molecular testing for
infectious diseases is the promise of earlier detection of certain
pathogens. The rapid detection of M. tuberculosis directly in
clinical specimens by PCR or other amplification-based methods is
quite likely to be cost-effective in the management of tuberculosis
[7]. Other examples of infectious disease that are amenable to
molecular diagnosis and for which management can be improved by this
technology include HSV encephalitis, Helicobacter pylori infection,
and neuroborreliosis caused by Borrelia burgdorferi. For HSV
encephalitis, detection of HSV in cerebrospinal fluid (CSF) can
direct specific therapy and eliminate other tests including brain
biopsy. Likewise, detection of H. pylori in gastric fluid can direct
therapy and obviate the need for endoscopy and biopsy. PCR detection
of B. burgdorferi in CSF is helpful in differentiating
neuroborreliosis from other chronic neurologic conditions and chronic
fatigue syndrome.
As discussed earlier, molecular tests may be used to predict disease
response to specific antimicrobial therapy. Detection of specific
resistance genes (mec A, van A) or point mutations resulting in
resistance has proven efficacious in managing disease. Molecular-
based viral load testing has become standard practice for patients
with chronic hepatitis and AIDS. Viral load testing and genotyping of
HCV are useful in determining the use of expensive therapy such as
interferon and can be used to justify decisions on extent and
duration of therapy. With AIDS, viral load determinations plus
resistance genotyping have been used to select among the various
protease inhibitor drugs available for treatment, improving patient
response and decreasing incidence of opportunistic infections.
Pharmacogenomics is the use of molecular-based tests to predict the
response to specific therapies and to monitor the response of the
disease to the agents administered. The best examples of
pharmacogenomics in infectious diseases are the use of viral load and
resistance genotyping to select and monitor antiviral therapy of AIDS
and chronic hepatitis[17,18]. This application improves disease
outcome; shortens length of hospital stay; reduces adverse events and
toxicity; and facilitates cost-effective therapy by avoiding
unnecessary expensive drugs, optimizing doses and timing, and
eliminating ineffective drugs.
Molecular strain typing of microorganisms is now well recognized as
an essential component of a comprehensive infection control program
that also involves the infection control department, the infectious
disease division, and pharmacy[10, 21]. Molecular techniques for
establishing presence or absence of clonality are effective in
tracking the spread of nosocomial infections and streamlining the
activities of the infection control program[21,23]. A comprehensive
infection control program uses active surveillance by both infection
control practitioners and the clinical microbiology laboratory to
identify clusters of infections with a common microbial phenotype
(same species and antimicrobial susceptibility profile). The isolates
are then characterized in the laboratory by using one of a number of
molecular typing methods (Table 4) to confirm or refute clonality.
Based on available epidemiologic and molecular data, the hospital
epidemiologist then develops an intervention strategy. Molecular
typing can shorten or prevent an epidemic[23] and reduce the number
and cost of nosocomial infections (Table 5)[10]. Hacek et al.[10]
analyzed the medical and economic benefits of an infection control
program that included routine determination of microbial clonality
and found that nosocomial infections were significantly decreased and
more than $4 million was saved over a 2-year period (Table 5).
The true financial impact of molecular testing will only be realized
when testing procedures are integrated into total disease assessment.
More expensive testing procedures may be justified if they reduce the
use of less-sensitive and less-specific tests and eliminate
unnecessary diagnostic procedures and ineffective therapies.
Dr. Pfaller is professor and director of the Molecular Epidemiology
and Fungus Testing Laboratory at the University of Iowa College of
Medicine and College of Public Health. His research focuses on the
epidemiology of nosocomial infections and antimicrobial-drug
resistance.
Address for correspondence: Michael Pfaller, Medical Microbiology
Division, C606 GH, Department of Pathology, University of Iowa
College of Medicine, Iowa City, Iowa 52242, USA; fax:319-356-4916; e-
mail: michael-pfaller@...
Table 1. FDA-approved molecular diagnostic tests for infectious
diseasea
Test Method Companyb
Chlamydia trachomatis detection PCRc
LCR
TMA
Hybrid capture Roche
Abbott
Gen-Probe
Digene
Neisseria gonorrhoeae detection LCR
Hybrid capture Abbott
Digene
C. trachomatis/N. gonorrhoeae
screening/detection Hybridization
SDR Gen-Probe
Becton-Dickinson
Mycobacterium tuberculosis detection PCR
TMA Roche
Gen-Probe
HPV screening Hybrid capture Digene
CMV Hybrid capture
NASBA Digene
Organon Teknika
Grp A strep detection Hybridization Gen-Probe
HIV quantitation PCR Roche
Gardnerella, T. vaginalis, and Candida Hybridization Becton-Dickinson
Culture confirmation for bacteria and fungi Hybridization Gen-Probe
a The table contains examples of commercially available methods and
is not intended to be all-inclusive. Websites of the principle
manufacturers are a useful source of the most up-to-date information.
b Companies: Digene, Silver Spring, MD; Chiron, Emeryville, CA;
Roche, Branchburg, NJ; Organon Teknika, Durham, NC; Murex/Abbott,
Abbott Park, IL; Gen-Probe, San Diego, CA; Abbott, Abbott Park, IL;
Becton-Dickinson, Cockeysville, MD.
c PCR = polymerase chain reaction; LCR = ligase chain reaction; TMA =
transcription-mediated amplification; SDR = strand displacement
reaction; NASBA = nucleic acid strand-based amplification.
Table 2. Noncommercial nucleic acid-based tests for clinically
important viral and bacterial pathogensa
Organism Specimen type Clinical indication
Epstein-Barr virus (EBV) Cerebrospinal fluid (CSF) EBV
lymphoproliferative disorder
Herpes simplex virus (HSV) types 1 and 2 CSF
Vitreous humor Encephalitis
Varicella-zoster virus (VZV) Various tissues VZV reactivation
JC virus CSF Progressive multifocal leukoencephalopathy
Enterovirus CSF Aseptic meningitis
Parvovirus B19 Amniotic fluid
Serum Hydrops fetalis
Anemia
Adenovirus Urine
Tissues
Blood Immunocompromised patients, transplant recipients
Ehrlichia Blood Human granulocytic and monocytic ehrlichiosis
Bordetella pertussis Nasopharyngeal aspirate Whooping cough
Legionella pneumophila Respiratory Atypical pneumonia
Chlamydia pneumoniae Respiratory Atypical pneumonia
Mycoplasma pneumoniae Respiratory Atypical pneumonia
Helicobacter pylori Gastric fluid
Stool Peptic ulcer disease
a All tests use polymerase chain reaction. The list is not all-
inclusive.
Table 3. Molecular methods for detecting antimicrobial resistancea
Organism(s) Antimicrobial agent(s) Gene Detection method
Staphylococci Methicillin mec Ab Standard DNA probe
Oxacillin Branched chain DNA probe
PCR
Enterococci Vancomycin van A, B, C, Dc Standard DNA probe
PCR
Enterobacteriaceae Beta-lactams blaTEMandblaSHVd Standard probe
Haemophilus influenzae PCR and RFLP
Neisseria gonorrhoeae PCR and sequencing
Enterobacteriaceae and gram-positive cocci Quinolones Point mutations
in gyr A, gyr B, par C and par E PCR and sequencing
Mycobacterium tuberuclosise Rifampin Point mutations in rpo B PCR and
SSCP
PCR and sequencing
Isoniazid Point mutations in kat G, inh A, and ahp C PCR and SSCP
Ethambutol Point mutations in emb B PCR and RFLP
Streptomycin Point mutations in rps L and rrs PCR and sequencing
Herpes virusesf Acyclovir and related drugs Mutations or deletions in
the TK gene PCR and sequencing
Foscarnet Point mutations in DNA polymerase gene PCR and sequencing
HIVg Nucleoside reverse transcriptase inhibitors Point mutations in
RT gene PCR and sequencing
PCR and LIPA
Protease inhibitors Point mutations in PROT gene PCR and sequencing
a Adapted from Pfaller[2].
b mecA encodes for the altered penicillin binding protein PBP2a';
phenotypic methods may require 48 hours incubation or more to detect
resistance and are less than 100% sensitive. Detection of mecA has
potential for clinical application in specific circumstances.
c Vancomycin resistance in enterococci may be related to one of four
distinct resistance genotypes of which vanA and vanB are most
important. Genotypic detection of resistance is useful in validation
of phenotypic methods.
d The genetic basis of resistance to beta-lactam antibiotics is
extremely complex. The blaTEM and blaSHV genes are the two most
common sets of plasmid encoded beta-lactamases. The presence of
either a blaTEM or blaSHV gene implies ampicillin resistance.
Variants of the blaTEM and blaSHV genes (extended spectrum beta-
lactamases) may also encode for resistance to a range of third-
generation cephalosporins and to monobactams.
e M. tuberculosis is very slow growing. Four weeks or more may be
required to obtain phenotypic susceptibility test results. Detection
of resistance genes in M. tuberculosis has potential for clinical
application in the short term.
f There are no phenotypic methods sufficiently practical for routine
clinical detection of resistance to antiviral agents. Genotypic
methods represent a practical method for routine detection of
antiviral resistance.
g Abbreviations not defined in text: RFLP, restriction fragment
length polymorphism; SSCP, single-stranded conformational
polymorphism; LIPA, line probe assay; TK, thymidine kinase; RT,
reverse transcriptase; PROT, protease.
Table 4. Genotypic methods for epidemiologic typing of
microorganismsa,b
Method Examples Comments
Plasmid analysis Staphylococci Plasmids may be digested with
restriction endonucleases
Enterobacteriaceae Only useful when organisms carry plasmids
Restriction endonuclease analysis of chromosomal DNA with
conventional electrophoresis Enterococci Large number of bandst
Staphylococcus aureus Difficult to interpret
Clostridium difficile Not amenable to computer analysis
Candida spp.
PFGE Enterobacteriaceae Fewer bands
Staphylococci Amenable to computer analysis
Enterococci Very broad application.
Candida spp.
Genome restriction fragment length polymorphism analysis: ribotyping,
insertion sequence probe fingerprinting Enterobacteriaceae Fewer
bands
Staphylococci Computer analysis
Pseudomonas aeruginosa Sequence-based profiles
Mycobacterium tuberculosis Automated
Candida spp.
PCR-based methods: repetitive elements PCR spacer typing, selective
amplification of genome restriction fragments, multilocus allelic
sequence-based typing Enterobacteriaceae Crude extracts and small
amounts of DNA may suffice
Acinetobacter spp.
Staphylococci
M. tuberculosis
HCV
Library probe genotypic hybridization schemes: multilocus probe dot-
blot patterns, high-density oligonucleotide patterns Burkholderia
cepacia Unambiguous yes-no result
S. aureus Less discrimination than other methods
M. tuberculosis Couple with DNA chip technology
a The table contains examples of available methods and applications
and is not intended to be all-inclusive.
b Adapted from Pfaller[2].
Table 5. Reduction in number and cost of nosocomial infections
through collaborative efforts of infection control, clinical
microbiology, and molecular typing laboratoriesa
Time period Nosocomial infection rate (%)b Reduction in total
infections (no.) Reduction in cost
(million $)
94 vs. 95 94 vs. 96 94 vs. 95 94 vs. 96
FY 1993 3.3
FY 1994 3.4
FY 1995 2.6 301 1.8
FY 1996 2.6 344 2.6
aAdapted from Hacek et al.([10].
bPercentage of patients with nosocomial infections