Chapter 11

Detection and Identification

of Microorganisms

Outline Restriction Fragment Length Polymorphism Analysis

Arbitrarily Primed PCR
SPECIMEN COLLECTION Amplified Fragment Length Polymorphism (AFLP) Assay
SAMPLE PREPARATION Interspersed Repetitive Elements
QUALITY ASSURANCE Internal Transcribed Spacer Elements
Controls spa Typing
Quality Control Multilocus Sequence Typing
Selection of Sequence Targets for Detection of Microorganisms Mass Spectrometry
MOLECULAR DETECTION OF MICROORGANISMS Comparison of Typing Methods
Bacteria
Respiratory Tract Pathogens
Urogenital Tract Pathogens
Viruses Objectives
Mass Spectrometry
Mycology 11.1 Name the organisms that are common targets for
Parasites molecular-based laboratory tests.
ANTIMICROBIAL AGENTS 11.2 Identify advantages and disadvantages of using
Resistance to Antimicrobial Agents molecular-based methods as compared with
Molecular Detection of Resistance traditional culture-based methods in the detection
Beta-Lactam Antibiotic Resistance and identification of microorganisms.
Glycopeptide Antibiotic Resistance 11.3 Differentiate between organisms for which
Antimicrobial Resistance in M. tuberculosis commercially available nucleic acid amplification
MOLECULAR EPIDEMIOLOGY tests exist and those for which “home-brew”
Molecular Strain Typing Methods for Epidemiological Studies polymerase chain reaction (PCR) is used.
Plasmid Analysis 11.4 List the genes involved in the emergence of
Pulsed-Field Gel Electrophoresis antimicrobial resistance that can be detected by
nucleic acid amplification methods.
301
302 Section III • Techniques in the Clinical Laboratory

11.5 Compare and contrast the molecular methods Additionally, molecular-based tests have been devel-
that are used to type bacterial strains in oped for organisms that are received in clinical laborato-
epidemiological investigations. ries in high volumes, such as Streptococcus pyogenes in
11.6 Explain the value of controls, in particular, throat swabs and Neisseria gonorrhoeae and Chlamydia
amplification controls, in ensuring the reliability of trachomatis in genital specimens. Furthermore, genes
PCR results. that confer resistance to antimicrobial agents are the
11.7 Interpret pulsed-field gel electrophoresis patterns targets of molecular-based methodologies, such as mecA,
to determine whether two isolates are related to which contributes to the resistance of Staphylococcus
or different from each other. aureus to oxacillin; vanA, vanB, and vanC, which give
Enterococcus resistance to vancomycin; tonB, which
confers resistance to carbapenems; and katG and inhA,
which mediate M. tuberculosis resistance to isoniazid.
Furthermore, mass spectrometry can directly identify
Microbiological applications for the clinical laboratory resistance factors such as expressed β-lactamases even
are increasingly based on the molecular characterization in the absence of antibiotics.1
of microorganisms and the development and evaluation Finally, characterization of DNA, RNA, and protein
of molecular-based laboratory tests of clinical specimens was developed to find and identify new organisms and
isolated in cultures. Another important application of to further characterize or classify known organisms,
molecular technology in the clinical microbiology lab- such as influenza virus.2 Nucleic acid sequence infor-
oratory is in the comparison of biochemically similar mation is used to reclassify bacterial organisms based
organisms in outbreak situations, known as molecular on 16S rRNA sequence homology, for epidemiological
epidemiology, to ascertain whether the isolates have a purposes, and to predict therapeutic efficacy. Mass spec-
common or independent source. Clinically important trometry is also being applied to the identification of
microorganisms include a range of life-forms, from microorganisms based on peptide profiles.
arthropods to prions, and although molecular-based The molecular methods used in the clinical micro-
methods have become routine in clinical microbiol- biology laboratory are the same as those that were
ogy, traditional culture and biochemical testing are still described previously for the identification of human
important for the detection and identification of a variety polymorphisms and those that will be discussed in sub-
of microorganisms. sequent chapters for the identification of genes involved
In contrast to the analysis of phenotypic traits (micro- in cancer and in inherited diseases. These include poly-
scopic and colonial morphologies, enzyme or pigment merase chain reaction (PCR)—traditional, real-time,
production, carbohydrate fermentation patterns), the and reverse transcriptase PCR, and DNA sequencing.
analyte for molecular testing is the genome, transcrip- Additional methods used in molecular epidemiology are
tome, or proteome of the microorganism. Bacteria, fungi, pulsed-field gel electrophoresis (PFGE), matrix-assisted
and parasites have DNA genomes, whereas viruses can laser desorption ionization (MALDI) spectrometry, and
have DNA or RNA genomes. Prions, which cause trans- other methods that are discussed in this chapter. The
missible encephalopathies such as Creutzfeldt–Jakob development of molecular-based methods has been suc-
disease, consist only of protein. cessful for some organisms but not yet for all organisms,
Microorganisms targeted by molecular-based lab- as discussed in this chapter.
oratory tests have been those that are difficult and/
or time-consuming to isolate, such as Mycobacterium
tuberculosis as well as other species of Mycobacterium; SPECIMEN COLLECTION
those that are hazardous with which to work in the
clinical laboratory, such as Histoplasma and Coccidioi- As with any clinical test, proper procedure is impor-
des; and those for which reliable laboratory tests were tant for collection and transport of specimens for infec-
lacking, such as hepatitis C virus (HCV) and human tious disease testing. Microbiological specimens may
immunodeficiency virus (HIV). require special handling to preserve the viability of the
 Chapter 11 • Detection and Identification of Microorganisms 303

target organism. Special collection systems have been

designed for the collection of strict anaerobes, viruses, TABLE 11.1 Specimen Transport Systems
and other fastidious organisms. Although viability is
not as critical for most molecular testing, the quality Type Examples
of nucleic acids may be compromised if the specimen Sterile Sterile cups, screw-capped tubes,
is improperly handled. DNA and especially RNA will containers stoppered tubes, Petri dishes
be damaged in lysed or nonviable cells. Due to the
sensitivity of molecular testing, it is also important to Swabs Calcium alginate swabs, Dacron
swabs, cotton swabs, nasopharyngeal-
avoid contamination that could yield false-positive urogenital swabs, swab transport system
results.
Collection techniques designed to avoid contami- Specialty Neisseria gonorrhoeae transport systems,
nation from the surrounding environment of adjacent systems Swab Extraction Tube System (SETS)
tissues apply to molecular testing, especially to those Proprietary Molecular testing, N. gonorrhoeae
tests that use amplification methods. Sampling must systems transport systems, STAR buffer42
include material from the original infection. The time
Anaerobic Starplex Anaerobic Transport system
and site of collection should be optimal for the likely
transport (Fisher), BBL Vacutainer Anaerobic
presence of the infectious agent. For example, Salmo- systems Specimen Collector
nella typhi is initially present in peripheral blood but not
in urine or stool until at least 2 weeks after infection. For Viral transport BD Cellmatics Viral Transport Pack, BBL
systems Viral Culturette
classical methods that include culturing of the agent, a
sufficient number of microorganisms must be obtained
for agar or liquid culture growth. For molecular testing,
however, minimum numbers (as few as 50 organisms)
can be detected successfully. The quantity of target
organisms, as well as the clinical implications, should be
taken into account when interpreting the significance of
positive results. Molecular detection can reveal infective
agents at levels below clinical significance. Conversely,
highly specific molecular methods may miss detection
of a variant organism.
Equipment and reagents used for specimen collection
are also important for molecular testing (Table 11.1).
Blood draws should go into the proper anticoagulant,
if one is to be used. Although wooden-shafted swabs
may be used for throat cultures, Dacron or calcium algi-
FIGURE 11.1 The Swab Extraction Tube System (SETS)
nate swabs with plastic shafts have been recommended
consists of a punctured inner tube that holds a swab and fits in
for collection of bacteria, viruses, and mycoplasma a capable outer tube. Under the force of centrifugation, liquid
from mucosal surfaces.3 The plastics are less adherent in the swab is forced through the inner tube and into the outer
to the microorganisms and will not interfere with PCR tube, where it can be stored.
reagents, with the exception of calcium alginate swabs
with aluminum shafts, which had been reported to affect
PCR amplification.4 Collection media or amplifica- Commercial testing kits supply an optimized collec-
tion system may influence positivity rates.5 Collection tion system for a particular test organism. The Clinical
methods such as Swab Extraction Tube System (SETS), and Laboratory Standards Institute has published docu-
sonication, and vortex have been designed for maximum ments addressing the requirements for transport devices
recovery of microorganisms from swabs by centrifuga- and quality control guidelines. The College of American
tion6 (Fig. 11.1). Pathologists requires documented procedures describing
304 Section III • Techniques in the Clinical Laboratory

specimen handling, collection, and transport in each preparation methods. Finally, if RNA is to be analyzed,
laboratory. inactivation or removal of RNases in the sample and in
all reagents and materials that come into contact with the
sample is important.
SAMPLE PREPARATION Any clinical specimen can be used as a source of
microorganism nucleic acid for analysis. Depending on
Isolating nucleic acids from microorganisms is similar the specimen, however, special preparation procedures
to isolating nucleic acids from human cells with only may be necessary to allow for optimal nucleic acid
a few additional considerations. First, depending on the isolation, amplification, and analysis. The presence of
microorganism, more rigorous lysis procedures may be inhibitors of DNA polymerase has been demonstrated in
required. Mycobacteria and fungi, in particular, have clinical samples; therefore, careful separation of nucleic
thick cell walls that are more difficult to lyse than those acid from other molecules present in the sample will
of other bacteria and parasites. Gram-positive bacteria ensure target amplification.7 When processing a whole-
has a thicker cell wall than gram-negative bacteria and blood specimen, it is important to remove hemoglobin
may require more rigorous cell lysis conditions. Myco- and other products of metabolized hemoglobin because
plasma, on the other hand, lacks a cell wall, and so care they can inhibit DNA polymerase and thus may prevent
must be taken with the sample to avoid spontaneous the amplification of nucleic acid in the sample, resulting
lysis of the cells and loss of nucleic acids. in a false-negative PCR result. Eukaryotic cells can be
used as a source of nucleic acid for organisms, primarily
viruses that infect these cells. In blood samples, white
Advanced Concepts blood cells are isolated from the red blood cells using
Ficoll-Hypaque and then lysed. Alternatively, whole
Biological safety is an important concern for clin-
blood is processed in automated DNA isolation systems,
ical microbiology. Because various collection,
which effectively remove hemoglobin and other con-
transport, and extraction systems inactivate organ-
taminating molecules. Serum and plasma (devoid of red
isms at different times, the technologist should
blood cells) are also used as sources of microorganism
follow the recommendations of the Centers for
nucleic acid.
Disease Control and Prevention (CDC) that call
Sputum is a source of nucleic acid from organisms
for universal precautions, treating all specimens as
that cause respiratory tract infections. Acidic poly-
if they were infectious throughout the extraction
saccharides present in sputum may inhibit DNA poly-
process. Updated guidelines are available from
merase and thus must be removed. Using a method or
the CDC for the handling of suspected bioterror-
an instrument that reliably separates DNA from other
ism material. Organisms such as smallpox must
cellular molecules is sufficient to remove the inhibitors.
be handled only in approved (level 4 contain-
Urine, when sent for nucleic acid isolation and amplifi-
ment) laboratories. Molecular testing has eased
cation, is treated similarly to cerebrospinal fluid; that is,
the requirements for laboratory culture. Methods
the specimen is centrifuged to concentrate the organisms
devised to replace the growth of cultures should
and then subjected to nucleic acid isolation procedures.
improve safety levels.
Inhibitors of DNA polymerase—nitrate, crystals, hemo-
globin, and beta-human chorionic gonadotropin—have
Second, the concentration of organisms within the clin- been demonstrated in urine as well.8
ical sample must be considered. Samples can be centri- The type of specimen used for molecular testing
fuged to concentrate the organisms within the fluid from will also affect extraction and yield of nucleic acid.
the milliliters of sample that are often received down to For example, viral nucleic acid from plasma is easier
volumes that are appropriate for molecular procedures. to isolate than nucleic acid from pathogenic Entero-
Third, inhibitors of enzymes used in molecular anal- coccus in stool specimens. Reagents and devices have
ysis may be present in clinical specimens; removal or been developed to combine collection and extraction of
inactivation of inhibitors might be included in specimen nucleic acid from difficult specimens; for example, stool
 Chapter 11 • Detection and Identification of Microorganisms 305

transport and recovery (STAR) buffer or the FTA paper a positive amplification control signal, whereas the
systems that inactivate infectious agents and adhere reagent blank should be negative for amplification. With
nucleic acids to magnetic beads or paper, respectively. a positive amplification control, lack of amplification
of the target can be more confidently interpreted as a
true negative result. Amplification controls are usually
QUALITY ASSURANCE housekeeping genes or those that are always present in
human or microbiological samples. Housekeeping genes
For any type of medical laboratory procedure, quality that are used as internal controls include prokaryotic
control is critical for ensuring the accuracy of patient genes, such as groEL, rpoB, recA, and gyrB, and eukary-
results. Ensuring the quality of the molecular methods is otic genes, such as β-actin, glyceraldehyde-3-phosphate,
equally important. Molecular testing sensitivity is rela- interferon-γ, extrinsic homologous control, human mito-
tively high, so even one molecule of target is a potential chondrial DNA, and peptidylprolyl isomerase A.9
template. Thus, the integrity of specimens, that is, spec- Internal controls are amplification controls that mon-
imens not contaminated by other organisms or with the itor particular steps of an amplification method. They
products of previous amplification procedures, is critical can be either homologous extrinsic, heterologous ex-
to avoid inaccurate results. Also, it is equally important trinsic, or heterologous intrinsic (Fig. 11.2). A homol-
to ensure that the lack of a product in an amplification ogous extrinsic control is a target-derived control with a
procedure is due to the absence of the target organism non-target-derived sequence insert. This control is added
and not the presence of inhibitors preventing the ampli- to every sample after nucleic acid extraction and before
fication of target sequences. amplification. The amplification of this control occurs
using the same primers as for the target, which is good
for ensuring that amplification occurs in the sample
Controls
but does not control for target nucleic acid degrada-
Control substances of known composition are used to tion during extraction. Heterologous extrinsic controls
monitor the reliability of the method and the input spec- are non-target-derived controls that are added to every
imen material. The incorporation of positive controls sample before nucleic acid extraction. This control will
shows that an assay system is functioning properly. A ensure that the extraction and amplification procedures
sensitivity control that is positive at the lower limit of were acceptable, but a second set of primers must also
detection demonstrates the sensitivity of qualitative be added to the reaction for the control to be amplified.
assays. Two positive controls, one at the lower limit and Using this control requires that the procedure be opti-
the other at the upper limit of detection, are run in quan- mized so that the amplification of the control does not
titative assays to test the dynamic range of the assay. interfere with the amplification of the target. Heterolo-
Reagent blank or contamination controls are critical for gous intrinsic controls are nontarget sequences naturally
monitoring reagents for contamination; the latter con- present in the sample, such as eukaryotic genes in a test
tains all of the assay reagents except target sequences and for microorganisms. In this case, human gene controls
should always be negative. For typing and other studies serve to ensure that human nucleic acid is present in the
that might include nontarget organisms, a negative tem- sample in addition to controlling for extraction and am-
plate control might also be included. This control will plification. Using this control requires that either two am-
detect the presence of the unwanted target(s), but should plification reactions be performed on the sample, one for
not react with the desired target. the control and the other for the target gene, or that the
With regard to amplification methods that are inter- amplification procedure be multiplexed, as long as there
preted by the presence or absence of product, false- is no interference with the amplification of the target.
negative results can occur due to amplification failure.
In order to rule out this type of false-negative result,
Quality Control
an amplification control aimed at a target that is always
present can be incorporated into an amplification assay. In a procedure that detects a microorganism, a positive
The negative template control sample should have result states that the organism is present in that sample,
306 Section III • Techniques in the Clinical Laboratory

Target

Target organism
Homologous extrinsic

Plasmid

Heterologous extrinsic

Nontarget
organism

Heterologous intrinsic

Host

FIGURE 11.2 Amplification controls and their relationships to the molecular target. Homologous extrinsic controls are a modi-
fied version of the target that maintain the target primer binding sites. The homologous intrinsic control may be smaller, larger, or
the same size as the target. Heterologous extrinsic controls are obtained from unrelated nontarget organisms and require primers
different from those of the target sequences. Heterologous intrinsic controls are similar to heterologous extrinsic controls, except
that heterologous intrinsic controls come from host sequences.

whereas a negative result indicates that the organism the laboratory. Second, amplification procedures may be
is not present (at amounts up to the detection limits of inhibited by substances present in the specimen. Hemo-
the assay). Although most false-positive test results can globin, lactoferrin, heparin and other anticoagulants,
be eliminated by preventing carryover contamination, sodium polyanethol sulfonate (anticoagulant used in
another source of false-positive test results that cannot blood culture media), and polyamines have been shown
be controlled in the laboratory is the presence of dead to inhibit nucleic acid amplification procedures.10 Atten-
or dying microorganisms in the sample of a patient tion to nucleic acid isolation procedures and ensuring
taking antimicrobial agents. In this situation, the nucleic optimal purification of nucleic acid from other compo-
acid–based tests will remain positive longer than culture nents of the specimen and extraction reagents will help
assays and thus may appear as a false positive. Repeat- minimize the presence and influence of inhibitors on the
ing the nucleic acid–based assay 3 to 6 weeks after anti- amplification reaction.11 As with all clinical tests, vali-
microbial therapy is more likely to yield a true-negative dation must be performed on new molecular-based tests
result. False-negative results may be more problematic that are brought into the laboratory. Controls must be
and arise when the target organism is present but the tested, and the sensitivity, specificity, and reproducibility
test result is negative. There are a few reasons for false- of the assay must be determined using reference mate-
negative results on a sample. First, the organism may rials.12 Proficiency testing of methods and competency
be present, but the nucleic acid was degraded during testing of personnel should be performed regularly. The
collection, transport, and/or extraction. This degradation Clinical and Laboratory Standards Institute, Associa-
can be prevented by proper specimen handling, effective tion for Molecular Pathology, and the Food and Drug
transport media, and inhibiting the activity of DNases Administration (FDA) have guidelines for molecular
and RNases that may be present in the sample and in methods in the laboratory.
 Chapter 11 • Detection and Identification of Microorganisms 307

Selection of Sequence Targets for Detection HIV or other retroviruses have variable sequences within
of Microorganisms the same species. Such variations may be informative in,
for instance, determining drug resistance or for epidemi-
Molecular methods are based on sequence hybridiza- ological information; however, not all types would be
tion or recognition using known nucleic acid sequences detected by a single sequence. The variable sequences
(primers or probes). These tests are limited by the choice may be included in the probe or primer areas to differen-
of target sequences for primer or probe hybridization. tiate between types. These type-specific probes/primers
The primary nucleotide sequence of many clinically are used in a confirmatory test after an initial test using
important microorganisms is available from the National probes or primers directed to a sequence shared by
Center for Biological Information (NCBI) or from pub- all types.
lished literature. The specificity of molecular methods In addition to their strain or species specificity, target
targeting these sequences depends on the primers or sequences must meet technical requirements for hybrid-
probes that must hybridize specifically to the chosen ization conditions. Primers should have similar anneal-
point in the genome of the microorganism. ing temperatures and yield amplicons of appropriate
Choosing a sequence target is critical for the specific- size. Probes must hybridize specifically under the con-
ity of a molecular test (Fig. 11.3). Many microorganisms ditions of the procedure. Sequence differences can be
share the same sequences in evolutionarily conserved distinguished using sequence-specific probes or primers.
genes. These sequences would not be used for detection Design of probe-based amplification or detec-
of specific strains as they are likely to cross-react over tion methods, includes decisions as to the length and
a range of organisms. Sequences unique to the target sequence structure of the probe, whether the probe is
organism are therefore selected. Some organisms such as DNA, RNA, or protein, and how the probe is labeled.
The source of the probe is also important, as probes
must be replenished and perform consistently over long-
term use. Probes are manufactured synthetically or bio-
A B C
logically by cloning. Synthetic oligonucleotides may be
Genome preferred for known sequences where high specificity
is required. Primer design includes the length and any
Target organism modifications of the primers and type of signal genera-
tion for quantitative PCR.
Many tests currently used in molecular microbiol-
Genome
ogy are supplied as commercially designed systems,
including prevalidated probes and/or primers. Several
Target organism
(variant) of these are FDA-approved or FDA-cleared methods
(http://www.fda.gov). Manufacturers of these commer-
Genome cial reagents specify requirements for quality assurance,
including controls and assay limitations. Each system
Other flora
must be validated in the testing laboratory on the type
of specimen used for clinical testing, including serum,
FIGURE 11.3 Selection of target sequences for a nucleic acid plasma, cerebrospinal and other body fluids, tissue, cul-
test. The genomes of three organisms—the test target, a variant tured cells, and organisms. In addition to the commercial
or different type of the test target, and another nontarget organ-
reagent sets, many professionals working in medical lab-
ism—are depicted. Sequence region A is not specific to the
oratories have developed in-house laboratory protocols
target organism and is, therefore, not an acceptable area for
probe or primer binding to detect the target. Sequences B and (laboratory-developed tests [LDTs]) for which primers
C are specific to the target. Sequence B is variable and can be are designed based on sequence information that has
used to detect and type the target, although some variants may been published; the reagents are bought separately, and
escape detection. Sequence C will detect variants of the target the procedures are developed and optimized within the
organism but cannot be used for determining the type. individual laboratory.
308 Section III • Techniques in the Clinical Laboratory

MOLECULAR DETECTION OF MICROORGANISMS following the amplification of the target (Fig. 11.4). Like
conventional PCR, qPCR is performed on nucleic acid
Molecular-based methods that have been used to detect extracted directly from clinical specimens, including
and identify bacteria include nucleic acid-based hybrid- viral, bacterial, and fungal pathogens.
ization and amplification procedures. Target detection is Designing a qPCR method requires selecting a target
accomplished by a variety of methods, including agarose gene unique to the specimen or specimen type for which
gel electrophoresis, amplification methods (PCR, TMA, primers and probes can be designed. The DNA-specific
loop-mediated isothermal amplification [LAMP]), dye, SYBR green, can be used in place of probes if
sequencing, immunoassays, western blots, and mass the amplicon is free of artifact, such as mis-primes or
spectrometry. primer dimers. The probe types most often used include
Real-time PCR, or quantitative PCR (qPCR), is fluorescent energy transfer hybridization or hydrolysis
used frequently for the detection of infectious agents probes.
because it provides a sensitive, safe closed-tube assay The requirement for probes in addition to primers
with quantitative information not available from conven- increases the complexity of the design process. Instru-
tional PCR or other “end-point” amplification methods. ment software and several websites offer computer
The quantitative capability of qPCR allows distinction programs that automatically design primers and probes
of subclinical levels of infection (qualitatively positive on submitted sequences. Commercial primer and probe
by conventional PCR) from higher levels with patho- sets are also available for purchase as reagent sets with
logical consequences. Furthermore, qPCR programs can optimized buffers and required reaction components. A
be designed to provide closed-tube sequence or typing variety of gene targets have been used for qPCR detec-
analysis by adding a melt-curve temperature program tion of a number of organisms. A list of examples of

0.08
Fluorescence (FZ/Back–F1)

0.07
BK
0.06
0.05
0.04
JC
0.03
0.02
0.01
0
55 60 65 70 75 80 85
Temperature (°C)
Fluorescence, d(FZ/Back–F1)dt

0.012
0.01 BK
JC
0.008 FIGURE 11.4 Melt-curve analysis of BK and JC
0.006 viruses. BK and JC are differentiated from one another
by differences in the Tm* of the probe specific for each
0.004
viral sequence. Fluorescence from double-stranded DNA
0.002 decreases with increasing temperature and DNA denatur-
0 ation to single strands (top panel). Instrument software
–0.002 will present a derivative of the fluorescence (bottom
60 62 64 66 68 70 72 74 76 78 80 panel) where the Tms (67°C to 68°C for BK and 73°C to
Temperature (°C) 74°C for JC) are observed as peaks. See Color Plate 9.
 Chapter 11 • Detection and Identification of Microorganisms 309

targets and probes is presented in a comprehensive Bacteria

review by Espy et al.13
Respiratory Tract Pathogens
Similar to standard PCR, useful genes for qPCR
methods include ribosomal RNA (rRNA), both 16S and Bacteria that cause respiratory tract disease are ubiqui-
23S, and housekeeping genes such as groEL, rpoB, recA, tous in the environment and are endemic (native to a
and gyrB. The 16S rRNA is a component of the small certain region or population group) even in higher socio-
subunit of the prokaryotic ribosome, and the 23S rRNA economic countries. Bacteria in the respiratory tract are
is a component of the large subunit of the prokaryotic easily transmitted by contact with infected respiratory
ribosome. Sequencing of the DNA region encoding secretions. Laboratory detection and identification of
16S rRNA (rDNA) is performed to determine the evo- these organisms by nonmolecular methods often lack
lutionary and genetic relatedness of microorganisms and sensitivity and/or are time-consuming. Because of their
has driven changes in microorganism nomenclature.14 importance in causing human disease, molecular-based
The rDNA that encodes the rRNA consists of alternat- assays that can detect and identify bacterial pathogens
ing regions of conserved sequences and sequences that directly in respiratory specimens have been developed
vary greatly from organism to organism. The conserved (see Table 11.2).
sequences encode the loops of the rRNA and can be Frequent testing targets include Bordetella, Legio-
used as a target to detect all or most bacteria. Sequences nella, Mycobacteria, Chlamydia, and Streptococcus
that have a great amount of heterogeneity encoded in species. Individual IVD and analyte-specific reagent
the stems of the rRNA can be used to detect a specific (ASR) systems have been marketed for individual testing
genus or species of bacteria. Ribosomal RNA was the from a variety of specimen sources. Multiplex tests are
original target of many bacterial molecular-based assays, also performed for screening or speciation.
but because of the instability and difficulty in analyzing Bordetella pertussis is a pathogen of the upper
RNA, current assays amplify and detect rDNA sequences respiratory tract that is the causative agent of whoop-
and proteins. ing cough. The organism is endemic worldwide and
Mass spectrometry of microbial proteins has been is transmitted via direct contact with infected respira-
applied to microbiological identification and epidemiol- tory secretions. Primer and probe ASR for B. pertus-
ogy.15,16 In MALDI technology, proteins are converted sis and Bordetella parapertussis detection by qPCR
into singly charged ions in an energy-absorbent matrix. targeting IS481 and IS1001, respectively, have been
For microbiological applications, the matrix is an available.
acidic compound such as sinapinic acid, or a-cyano-4- Legionella pneumophila is the cause of Legionnaires’
hydroxycinnamic acid (CHCA) dissolved in an organic disease, an infection of the lower respiratory tract
mixture of ethanol or methanol and a strong acid. The that was first described in men attending an American
solvents penetrate cell walls and membranes, extract- Legion convention in Philadelphia in 1976. Since their
ing the intracellular proteins. Some organisms can be first identification, Legionella species have been found
spotted directly from a single colony and covered with in water, both in the environment as well as in air condi-
matrix. Initial extraction in formic acid is required for tioners and hot water tanks in various types of buildings.
reproducible identification of gram-positive organisms Legionella species infections range from asymptomatic
and fungi.17 to fatal and are the third most common cause of com-
Peptide databases are the central determinant in munity-acquired pneumonias.18 PCR tests for Legionella
mass spec. These profiles, also called protein mass have targeted the macrophage infectivity potentiator
fingerprints, are maintained by instrument manufac- (mip) gene and 16S and 5S rRNA genes.
turers and also available as open-source options. In M. tuberculosis is an important cause of respiratory
vitro diagnostics (IVD) versions of databases of over tract infections causing significant levels of morbidity
200 profiles are used to identify species and strains. and mortality. The diagnosis of tuberculosis (TB) may
Several factors, including culture, sample preparation, take prolonged periods, during which time infections can
and instrument technology, influence the informativity spread. The genome of M. tuberculosis has 4.4 million
of profiles produced. base pairs (bp) with about 4,000 genes. The genomes of
310 Section III • Techniques in the Clinical Laboratory

TABLE 11.2 Typical Respiratory Tract Organisms Targeted by Molecular-Based Detection Methods73,74

Organism Specimen Source Gene Target Traditional Diagnostic Methods

Mycoplasma Bronchoalveolar lavage 16S rRNA Culture

pneumoniae 16S rDNA Serology
Species-specific protein gene
P1 adhesion gene

Chlamydophila Respiratory Cloned Pst I fragment Culture

pneumoniae Throat 16S rRNA
Atherosclerotic lesions MOMP

Legionella Deep respiratory secretions 5S rRNA mip gene Culture

Serum 16S rRNA Antigen detection
Buffy coat
Urine

Bordetella Nasopharyngeal IS 481 Culture

pertussis Adenylate cyclase gene Direct fluorescent antibody
Porin gene
Pertussis toxin promoter region

Streptococcus Blood DNA polymerase gene Culture

pneumoniae Cerebrospinal fluid plyA (pneumolysin)
Serum lytA (autolysin)
Sputum pbp2a (penicillin-binding protein)
pbp2b
pspA (pneumococcal surface protein)

Mycobacterium Sputum 16S rRNA Culture

tuberculosis Bronchoalveolar lavage
Bronchial washings
Gastric aspirates

different isolates of M. tuberculosis do not vary to any systems improved the detection rate of mycobacteria to
great extent, and most variation is due to the movement a few days, depending on the organism load.
of insertion elements rather than to point mutations.19 Nucleic acid amplification methodologies can detect
For many years, tuberculosis was detected from M. tuberculosis directly in a clinical specimen with
mycobacterial smears and culture. Whereas a fluoro- reliable sensitivity and specificity. PCR tests target-
chrome stain has increased sensitivity compared with the ing the species-specific sequences, such as IS6110 and
Kinyoun and Ziehl–Neelsen stains for detecting myco- 16S rRNA, allow detection of M. tuberculosis from
bacteria directly in clinical specimens. The sensitivity of fresh, frozen, or fixed tissue. PCR-positive samples are
smears in general for mycobacteria varies. At least 104 hybridized with genus-specific and species/complex-
organisms/mL are required in order to see mycobacteria specific probes. qPCR assays have also been developed
in a smear, and even then, not all of those smears read as for M. tuberculosis detection with primers and probes
positive. Cultures for M. tuberculosis are more sensitive targeting rRNA internal transcribed spacer (ITS) ele-
than smears and are able to detect 101 to 102 organisms/ ments in M. tuberculosis.
mL of specimen; however, they take time due to the slow Mycoplasma pneumoniae has been subjected to ampli-
in vitro growth of the organism. Liquid-based culture fication techniques and other characterizing methods
 Chapter 11 • Detection and Identification of Microorganisms 311

such as multilocus variable-number tandem-repeat molecular methods are so well characterized for these
(VNTR) analysis, multilocus sequence typing, and two organisms that they are used almost exclusively
matrix-assisted laser desorption ionization time-of-flight in the detection of the nucleic acid of N. gonorrhoeae
mass spectrometry (MALDI-TOF MS).20 and C. trachomatis. Other sexually transmitted bacte-
MALDI libraries of 50 to over 300 Mycobacterium ria are considered good targets for the development of
peptide spectra are offered by at least one manufacturer. molecular-based methods because traditional labora-
Due to the structure of their cell walls, mycobacteria tory methods of detection and identification for these
require special preparation for mass spectrometry testing. organisms either lack sensitivity or are time-consuming.
The bacteria are lysed by bead beating or in boiling Table 11.3 summarizes the molecular-based tests that
water (also a safety measure), extracted with ethanol, have been described for the bacteria that cause genital
dried, and resuspended in formic acid and acetonitrile tract infections.
for analysis. Several studies have shown favorable com- Cultures for N. gonorrhoeae and C. trachomatis
parison of MALDI-TOF with conventional methods. have been considered the gold standard, but nucleic
Chlamydophila pneumoniae is an obligate intracel- acid amplification assays have the advantages of
lular pathogen that causes 10% of community-acquired being rapid, and testing can be batched and automated,
pneumonias and has been implicated in atherosclerosis resulting in further savings for the laboratory. The first
and coronary artery disease. Despite the problems with molecular-based assay available for N. gonorrhoeae and
the development, implementation, and interpretation of C. trachomatis was a nonamplification-based nucleic
molecular-based assays for M. pneumoniae, L. pneu- acid hybridization method that detected the rRNA with
mophila, B. pertussis, and C. pneumoniae individually, an acridinium-labeled single-stranded DNA probe. DNA
multiplex nucleic acid amplification tests offer sensitive, probes have comparable sensitivities and specificities
specific, and rapid detection.21,22 to culture methods. Adding enhanced signal amplifica-
Streptococcus pneumoniae is a major cause of tion to the probe methods increased sensitivity. Numer-
community-acquired pneumonia and is also a common ous nucleic acid amplification assays are available that
cause of bacteremia, sepsis, otitis media, and meningi- target N. gonorrhoeae and C. trachomatis. These include
tis. Molecular-based tests targeting S. pneumoniae have target amplification assays, strand displacement ampli-
attempted to detect S. pneumoniae in various clinical fication, and transcription-mediated amplification. The
samples by targeting a variety of genes (see Table 11.2). nucleic acid amplification assays are performed on ure-
Although PCR is specific for S. pneumoniae, the clini- thral or cervical swabs, urine, and, in some cases, on
cal significance of a positive PCR assay is questionable transport vials that are used to collect cervical cells for
because a significant portion of the population (especially Papanicolaou smears with good sensitivity and spec-
children) is colonized with the organism, and PCR cannot ificity. Although molecular-based assays are the main
discern between colonization and infection.23,24 Sequenc- methods for detection of N. gonorrhoeae, C. tracho-
ing of 16S rRNA also does not allow enough discrimina- matis cultures may still be performed in conjunction
tion among alpha-hemolytic Streptococci species because with molecular-based assays in cases of suspected child
they share more than 99% sequence homology. Even abuse. Another consideration is laboratory testing to
MALDI-TOF identification S. pneumoniae is limited by confirm the cure of an infection. As with any infectious
database discrimination between closely related species. organism and molecular-based test, the nucleic acid is
Improvements in sample preparation methods, the use of detectable in a clinical sample whether the organism is
peak analysis, and updated databases have been applied dead or alive. It is recommended that a sample should
to distinguishing these species.25,26 not be taken for 3 to 4 weeks after treatment to confirm
treatment efficacy. Collection of samples and testing too
soon after treatment will result in positive results after
Urogenital Tract Pathogens
cultures on the same specimen have become negative.
Neisseria gonorrhoeae and Chlamydia trachomatis The spirochete T. pallidum subspecies pallidum is
were among the first organisms to be targeted for detec- the causative agent of syphilis, a sexually transmitted
tion in clinical specimens by molecular methods. The disease that results in the formation of a chancre at the
312 Section III • Techniques in the Clinical Laboratory

TABLE 11.3 Typical Genital Tract Organisms Targeted by Molecular-Based Detection Methods75

Organism Specimen Sources Traditional Diagnostic Methods Gene Target

Treponema Genital ulcers Serological (indirect and direct) TpN44.5a

pallidum Blood Direct antigen detection (dark field, TpN19
Brain tissue direct fluorescent antibody [DFA]) TpN39
Cerebrospinal fluid p01A
Amniotic fluid TpN47
Placenta 16S rRNA
Umbilical cord polA
Fetal tissue
Serum

Mycoplasma Urine Culture MgPa (adhesion gene)

genitalium Urethral rDNA gene
Vaginal
Cervical

Mycoplasma Genital tract Culture 16S rRNA

hominis Amniotic fluid

Ureaplasma Genital tract Culture 16S rRNA

urealyticum Amniotic fluid Urease gene

Haemophilus Gram stain 1.1 kb target

ducreyi Culture groEL gene
Serological Intergenic spacer between 16S and 23S rDNA
p27
16S rDNA gene

Neisseria Urine Culture omp III gene

gonorrhoeae Urethral opa gene
Cervical Cytosine DNA methyltransferase gene
Thin preparation vials cPPB gene
Site-specific recombinase gene

Chlamydia Urine Culture MOMP

trachomatis Urethral Enzyme immunoassay 16S RNA
Cervical DFA
Thin preparation vials
Conjunctiva

site of inoculation (primary syphilis). If left untreated, (rapid plasma reagin [RPR] and venereal disease
the organism disseminates through the body, damag- research laboratory [VDRL] tests) initially for the pres-
ing tissues. The patient may progress into the other ence of antibodies against cardiolipin (a normal compo-
stages of disease: secondary syphilis (disseminated nent of host membranes) and confirmed by testing for the
rash), latent syphilis (asymptomatic period), and ter- presence of antibodies against T. pallidum by enzyme
tiary syphilis (central nervous system and cardiovascular immunoassay to confirm infection. T. pallidum cannot
manifestations). be grown in vitro. Laboratories have adopted hemag-
Laboratory diagnosis of syphilis is limited to sero- glutination assays and enzyme immunoassays (EIAs)
logical testing, in which patients are typically screened to screen patients for syphilis and fluorescent antibody
 Chapter 11 • Detection and Identification of Microorganisms 313

absorption, particle agglutination assays, and the RPR the same time. Since this first description of the multi-
to monitor the effectiveness of treatment and to detect plex assay, other groups have used multiplex methods
reinfection.27,28 RPR and VDRL are limited in that, when to confirm the sensitivity of the assay for the targeted
reactive, they are not specific for syphilis, and the sensi- organisms and to examine the prevalence of these organ-
tivity of these tests in very early and late syphilis is low. isms in various geographic areas in different years.30,31
The serological tests that detect T. pallidum antibodies
are limited by their inability to differentiate between
Viruses
current and past infections. The RPR test, though, can
be used to detect reinfection because titers of anticardi- Evidence for virus infection has been detected by testing
olipin antibodies will decrease to nonreactive following for antibodies against the virus, by measuring the pres-
successful treatment of the organism and increase again ence or absence of viral antigens, or by detecting the
with reinfection. growth of a virus in a culture system. Although some of
Several PCR assays have been developed and tested these methods are well established for certain viruses,
for the direct detection of T. pallidum DNA in genital they all have major disadvantages.
ulcers, blood, brain tissue, cerebrospinal fluid, serum, Antibody detection is an indirect method of diag-
and other samples, with varying sensitivities. Amplifica- nosis. The host immune response has to be stimulated
tion of the T. pallidum DNA polymerase I gene (polA) by the virus to produce antibodies. In the case of an
is highly accurate when tested on genital, anogenital, immunodeficient patient, the lack of antibodies due to
or oral ulcers. PCR assays for T. pallidum that require host factors might be interpreted as a lack of the virus.
less than 1 mL of blood have accuracy comparable to Furthermore, antibodies are a retrospective indication
serology. of the infection. To interpret antibody testing with the
Mycoplasma hominis, Mycoplasma genitalium, and most confidence, paired sera should be collected, with
Ureaplasma urealyticum cause nongonococcal ure- one sample collected during the acute phase of the infec-
thritis. The mycoplasmas, as discussed previously for tion and the other collected as the patient is recovering.
M. pneumoniae, are the smallest free-living, self- The titers of antibodies are measured in both samples.
replicating organisms known. M. genitalium has the A fourfold or greater rise in titer level from the acute
smallest genome and thus was one of the first organisms sample to the convalescent sample indicates the pres-
to have its genome fully sequenced.29 M. genitalium ence of the virus during the acute stage. Detecting IgM
culture methods were labor intensive and not widely antibodies, in particular, during an acute infection is the
available. best evidence for the presence of a virus. But in patients
PCR assays were developed targeting the adhesion in the very early stages of infection, IgM titers may be
gene (MgPa) or the rDNA gene of M. genitalium. PCR below detection limits. During this “window” period,
was vital in establishing M. genitalium as an impor- the patient is infected and infectious.
tant genital tract pathogen, and laboratory-developed Antigen detection testing is available in the clinical
PCR with hybridization is used for clinical detection laboratory only for some viruses. Assays that measure
of M. genitalium as well as M. hominis and U. urea- viral antigens are available more often for respiratory
lyticum detection. Assay development was at one time syncytial virus (RSV), influenza virus, rotavirus, HSV,
hindered due to the absence of a reliable gold standard cytomegalovirus (CMV), and hepatitis B virus (HBV).
for comparison and especially in the absence of clini- Viral antigens are detected by enzyme immunoassays or
cal symptoms. Thus, the clinical significance of PCR- direct immunofluorescent assays.
positive specimens was difficult to interpret. Genital tract The classical method for detection and identifica-
specimens have been the target for the development of tion of viruses in body fluids is tissue or cell culture.
multiplex assays in which the presence of nucleic acid of Monolayers of host cells are grown in vitro, the patient’s
multiple organisms can be determined from one speci- specimen is inoculated onto the cells, and changes in
men in a single tube. The organisms causing genital tract the cells due to viral infection, called cytopathic effect,
infections often overlap in their symptoms, or infections are observed microscopically by the technologist. The
can be caused by the presence of multiple organisms at identity of the virus is confirmed using fluorescently
314 Section III • Techniques in the Clinical Laboratory

labeled monoclonal antibodies. Culture has been the

gold standard for many viruses, in particular adenovirus, TABLE 11.4 Genomes of Human Viruses
enteroviruses, CMV, influenza, and HSV, but it is not Double-Stranded DNA Viruses Adenovirus
applicable to other viruses, such as the hepatitis viruses, BK virus
because these viruses do not grow well in culture Cytomegalovirus
systems. Another disadvantage to viral culture is the Epstein–Barr virus
amount of time required before viral growth is detect- Hepatitis B virus
Herpes simplex virus 1
able. Although the shell vial system, where the sample Herpes simplex virus 2
is centrifuged onto a cell monolayer and tested with anti- Human papillomavirus
viral antibodies, decreased detection time, several days JC virus
to weeks, depending on the virus, can pass before detec- Molluscum virus
tion of cytopathic effect.32 Furthermore, some viruses do Rotavirus
Vaccinia virus
not produce a cytopathic effect on infecting cells, or the Varicella-zoster virus
cytopathic effect that is produced is subtle and easily
missed. In these cases, the cultures will be reported as Single-Stranded DNA Virus Parvovirus
a false-negative result. Another disadvantage of using
Double-Stranded RNA Virus Rotavirus
viral cultures to detect and identify viral infections is
that the specimen must be collected in the acute phase Single-Stranded RNA Viruses Colorado tick fever virus
of the disease, that is, in the first 5 days of the illness, Coronavirus*
Coxsackie virus*
after which the amount of virus in body fluids decreases
Dengue virus*
significantly and may result in false-negative cultures. Ebola virus†
Nucleic acid amplification assays have become indis- Echovirus*
pensable in the clinical virology laboratory. Molecular Hepatitis A virus*
methods are well suited to targeting the various con- Hepatitis C virus
Hepatitis D virus
figurations of nucleic acids found in human pathogenic
Hepatitis E virus
viruses (Table 11.4). Target amplification assays such as Human T-cell leukemia virus‡
PCR, reverse transcriptase PCR (RT-PCR), quantitative Human immunodeficiency virus‡
PCR (qPCR), and transcription-mediated amplification Influenza virus
(TMA) as well as signal amplification assays such as Measles virus†
Mumps virus†
branched DNA (bDNA) amplification and hybrid capture
Norwalk virus, norovirus
are used in the clinical virology laboratory to diagnose Parainfluenza virus
or monitor viral infections. Table 11.5 summarizes the Poliovirus*
viruses for which nucleic acid amplification assays have Rabies virus
been developed along with the type of amplification pro- Respiratory syncytial virus
Rhinovirus*
cedure, the targeted genes, and clinical utility.
Rubella virus*
Molecular-based tests for HIV, Epstein–Barr virus Yellow fever virus*
(EBV), human papillomavirus (HPV), HCV, and BK/JC
viruses are frequently used in clinical laboratories. *Positive RNA; directly translated.
Human immunodeficiency virus (HIV) is an RNA †

‡
Negative RNA; complementary to the translated strand.
Retroviral replication requires a DNA intermediate.
virus that makes a DNA copy of genomic RNA using its
own virally encoded reverse transcriptase. There are two
types of HIV: HIV type 1, or HIV-1, and HIV type 2, or virus that are found in different locations in the world.
HIV-2. HIV-2 is a minor isolate found mainly in West There are four HIV subtypes, M (Major), O (Outlier),
Africa and is less pathogenic than HIV-1, causing more N (non-M and Non-O), and P. HIV group M causes 95%
latent infections. New strains continue to evolve. of the infections due to HIV around the world. Group
HIV rapidly mutates and recombines so that multi- M is further divided into eight clades (A, B, C, D, F,
ple groups then divide into “clades,” or subtypes, of the G, H, and J). Group M, clade B, is found most often in
 Chapter 11 • Detection and Identification of Microorganisms 315

TABLE 11.5 Nucleic Acid Amplification (NAA) Tests for Viruses

NAA Dynamic Range/

Virus Methodology Amplified Target Sensitivity Clinical Utility

Human PCR gag gene; 155 bp (HIV-1 400–750,000 copies/mL Viral quantitation
immunodeficiency RT-PCR NASBA group M [subtypes A-H], (standard) Disease prognosis
virus bDNA not HIV-2 or HIV-1 group O) 50–100,000 copies/mL Treatment monitoring
gag (similar to Amplicor) (ultrasensitive)
HIV-1 groups M, O, and N 176,000–3,470,000
pol; subtypes of group M copies/mL
(subtypes A–G, but not 75–500,000 copies/mL
group O)

Cytomegalovirus Hybrid capture Immediate-early antigen 1 1,400–600,000 Detect CMV DNA in organ
(CMV) PCR Major immediate-early copies/mL transplant and AIDS
NASBA qPCR antigen 400–50,000 copies/mL patients and congenitally
Major capsid Glycoproteins B and H infected infants
protein Detect HSV when Viral load determinations
asymptomatic or when
cultures are negative

Herpes simplex PCR Thymidine kinase Diagnosis of HSV

virus-1 and -2 qPCR DNA polymerase encephalitis and neonatal
(HSV) DNA-binding protein infections
Glycoproteins gb, gc, gd,
and gg

Epstein–Barr virus PCR EBNA1 100–1010 copies/mL EBV-associated

(EBV) qPCR LMP-1 malignancies
Detect EBV in asymptomatic
immunocompromised hosts

Human Hybrid capture L1 or E1 open reading frames 105 copies/mL Detection of HPV in
papillomavirus PCR endocervical swabs
(HPV) qPCR Differentiation of low-risk
and high-risk types
Monitoring women with
abnormal Pap smears

Hepatitis B virus PCR 1,000–40,000 copies/mL Prognosis and monitoring

bDNA 0.7–5,000 meq/mL of antiviral treatment
Hybrid capture 142,000–1,700,000,000 response
copies/mL (standard)
4,700–56,000,000
copies/mL (ultrasensitive)

Parvovirus B19 PCR Diagnosis of infections

Respiratory RT-PCR Fusion glycoprotein (F) gene Detection of RSV

syncytial virus Nucleoprotein (N) gene Differentiate between
(RSV) subgroups A and B

Continued on following page

316 Section III • Techniques in the Clinical Laboratory

TABLE 11.5 Nucleic Acid Amplification (NAA) Tests for Viruses (Continued)

NAA Dynamic Range/

Virus Methodology Amplified Target Sensitivity Clinical Utility

Parainfluenza RT-PCR Hemagglutinin- Epidemiology

viruses neuraminidase conserved
regions
5’ noncoding region of F gene

Influenza viruses RT-PCR Conserved matrix (M) genes Diagnose infections

(influenza A and B) Characterize isolates
Nucleocapsid protein Can be type- and
(influenza A) subtype-specific
NS1 gene (influenza B)

Metapneumovirus RT-PCR Fusion (F) gene

RNA polymerase (L) gene

Coronavirus RT-PCR RNA polymerase gene Used to detect and

Nucleoprotein gene characterize the SARS virus

Norwalk virus RT-PCR RNA polymerase gene

Rotavirus RT-PCR VP7 gene

VP4 gene

Hepatitis C virus RT-PCR 5’ untranslated region (UTR) 600–800,000 IU/mL

bDNA 5’ UTR and core protein gene 3,200–40,000,000
copies/mL

West Nile virus RT-PCR Variety of gene targets based 0.1–1 PFU Used for diagnosis and
NASBA on genome of type strain surveillance
NY99
0.01 PFU

Rubella virus RT-PCR Surface glycoprotein, E1, gene Variable; sensitivity of Primarily for fetal diagnosis
3–10 copies is best Used for diagnosis when
serum is not available
May be used to confirm
positive serological results

Mumps virus RT-PCR Hemagglutinin, Differentiate strains

neuraminidase, P, SH, and F
genes

Measles virus RT-PCR M, H, F, N When culture is not

practical or genotyping is
required for diagnosis of
MIBE or SSPE
Differentiation of vaccine
and wild-type strains
 Chapter 11 • Detection and Identification of Microorganisms 317

TABLE 11.5 Nucleic Acid Amplification (NAA) Tests for Viruses (Continued)

NAA Dynamic Range/

Virus Methodology Amplified Target Sensitivity Clinical Utility

Enteroviruses RT-PCR Conserved 5’ nontranslated Performed on CSF to rule

(group A and B region out enteroviral meningitis
Coxsackieviruses,
echoviruses, and
others)

BK virus PCR Large T protein Diagnosis of BK virus

(polyomavirus) nephritis

MIBE, Measles inclusion body encephalitis; PFU, plaque-forming units; SSPE, subacute sclerosing panencephalitis.

the United States and Europe. Group O HIV is found The amount of HIV or viral load is used as a marker
primarily in West Africa, and group N is found in Cam- for disease prognosis as well as to track the efficacy of
eroon. To infect host cells, the HIV surface molecules antiretroviral therapy. The goal of antiretroviral therapy
gp120 and gp41 interact with CD4, a molecule that is is a viral load below 50 copies/mL of blood, where it
expressed primarily on the surface of helper T lympho- is undetectable by most methods. Patients who maintain
cytes but is also found on macrophages, dendritic cells, viral levels at fewer than 10,000 copies/mL in the early
and other antigen-presenting cells. Chemokine receptors, stages of the infection are at decreased risk of progres-
in particular CCR5, on dendritic/Langerhans’ cells and sion to acute immunodeficiency syndrome (AIDS).33
macrophages/monocytes, and CXCR4 on CD4+ T cells Patients who are effectively treated with antiretrovi-
form a complex with CD4 on the cell surface and ral therapy will have a significant reduction in viral load
also engage gp120. After attachment to host cells via 1 week after the initiation of therapy. The lack of a sig-
CD4-gp120 binding, the virus enters the cell, where nificant decrease in viral load during this time indicates
reverse transcriptase makes cDNA from viral RNA. The the lack of efficacy. Highly active antiretroviral therapy
cDNA integrates into the host DNA, where it either per- (HAART), consisting of two reverse transcriptase
sists in a stage of latency as a provirus or is replicated inhibitors combined with a protease inhibitor or a non-
actively. Transcription and translation of viral peptides, nucleoside reverse transcriptase inhibitor, reduced viral
as well as production of viral RNA, are performed loads below the detection limits of even ultrasensitive
by cellular components under the direction of virally assays.34 In patients receiving HAART, 2 log10 decreases
encoded regulatory proteins (i.e., tat, rev, nef, and vpr). in viral load have been documented. Viral load testing is
HIV infection is identified as antibodies specific for performed in conjunction with determining CD4 counts.
HIV in an EIA and by confirming the specificity of In general, but not always, viral load and CD4 counts
detected antibodies for HIV products in a western blot or are inversely proportional; that is, the higher the viral
a qualitative RNA probe assay. The antigens used in the load, the lower the CD4 count.
western blot tests may differ depending on the country HIV in patient samples can also be detected by
of origin. For infants who have maternal IgG and for PCR of integrated DNA, nucleic acid sequence–based
patients suspected of incubating HIV in whom antibody amplification (NASBA), and bDNA (see Table 11.5).
tests are negative, antigen detection tests measure the Table 11.6 compares the advantages and disadvan-
amount of HIV p24 antigen. Quantitative nucleic acid tages of each of these assays for determining HIV viral
amplification assays are performed after an HIV diag- load. Viral load should be determined before therapy is
nosis to determine how actively the virus is replicating started, 2 to 8 weeks after therapy initiation to see the
(viral load), when to start antiretroviral therapy, and initial response, and then every 3 to 4 months to assess
when to monitor the efficacy of treatment. therapeutic effectiveness.
318 Section III • Techniques in the Clinical Laboratory

TABLE 11.6 Advantages and Disadvantages TABLE 11.7 Test Performance Features
of the Nucleic Acid Amplification Methods for Viral Load Measurement
for HIV Viral Loads
Characteristic Description
Test Advantages Disadvantages
Sensitivity Lowest level detected at least 95% of
bDNA High throughput No internal control the time
Broad dynamic range False-positive
Applicable for group results reported Accuracy Closeness of measured value to a
M subtypes A–G standard or known value

Amplicor Internal control Limited dynamic Precision Reproducibility of independently

RT-PCR Good specificity range determined test results

NASBA Broad dynamic range Does not detect all Specificity Negative samples are always negative,
Performed on many non-B subtypes and positive results are true positives
specimen types and
Linearity A serial dilution of standard curve
volumes
closely approximates a straight line

Flexibility Accuracy of measurement of virus

Results among methods are increasingly comparable, regardless of sequence variations
such as viral loads determined by bDNA and RT-qPCR.35
Even so, it is still recommended that the same method to
determine viral loads is used when monitoring patients for viral load testing. It is established by the calibra-
over time. Results are expressed consistently as inte- tion of assays to a common standard. Quantitative tests
gers (copies/mL or IU/mL), log-transformed results (log must also have accuracy that is a true measure of the
IU/mL), scientific notation (a × 10b IU/mL), or a com- viral level over a range of values. The viral load levels
bination of these. CDC guidelines recommend report- established by the DHHS and the International AIDS
ing in integers and log-transformed copies. Quantitative Society for the initiation of therapy must be consistently
HIV-1 RNA testing in plasma has been the standard for identified in independent laboratories as accurately as
monitoring drug therapy and HIV disease progression. possible. Quantitative PCR methods offer linear mea-
The Multicenter AIDS Cohort Study, an ongoing project surement over a wider range than other methods, which
monitoring the clinical histories of treated and untreated precludes the requirement for dilution of high-titer spec-
HIV-infected men, has described laboratory mea- imens before analysis. The precision or reproducibility
surements of viral load and clinical disease. The U.S. of the test used is also important for establishing sta-
Department of Health and Human Services (DHHS) rec- tistically significant differences in the viral load over
ommends an objective of maximal suppression of viral a serial testing period. The DHHS defines a minimally
replication down to undetectable levels by sensitive significant change as a threefold increase or decrease in
analysis. viral load/mL plasma. And the high specificity of a test
The first commercially produced tests could detect gives confidence that a positive result is truly positive.
viral loads down to 400 to 500 copies/mL plasma; All subtypes of virus should be detected with equal effi-
however, suppression to fewer than 20 copies/mL plasma ciency to avoid under- or overestimating viral loads of
was associated with longer response to therapy than certain subgroups.
suppression below 500 copies/mL, which emphasizes HIV RNA in the same patient will not change much
the importance of a highly sensitive assay with a very over time (approximately 0.3 log10 unit) as long as the
low limit of detection for optimal treatment strategy and patient is clinically stable and antiretroviral therapy has
patient care. In addition to sensitivity, all test methods, not begun or changed. In order to be clinically relevant,
including HIV tests, should have certain characteris- viral load changes from one determination to another
tics (Table 11.7). Accuracy is an important requirement must vary by threefold (0.5 log10 unit). HIV-positive
 Chapter 11 • Detection and Identification of Microorganisms 319

patients may experience transient increases in viral loads Mutations found by genotyping are generally divided
when they have other infections or receive vaccinations, into two groups: primary resistance mutations and
but levels will return to baseline within a month. Profi- secondary resistance mutations. Primary resistance
ciency testing is available from the College of American mutations are those that are specific for a particular
Pathologists (CAP; Northfield, IL) and the CDC. The drug, reduce the susceptibility of the virus to that drug,
World Health Organization (WHO) has an HIV-1 RNA and appear in the viral genome shortly after treatment
reference standard. with that agent has begun. The mutated enzyme is gen-
Error-prone replication of HIV by its reverse tran- erally not as active as the normal enzyme, so viral repli-
scriptase and recombination between co-infecting strains cation is decreased, but it still occurs. As treatment with
generates new HIV sequences, which can affect the anti- the drug continues, secondary or compensatory muta-
viral drugs, including protease inhibitors, nucleoside tions occur that try to recover the ability of the virus to
analogs, reverse transcriptase inhibitors, and inhibitors replicate at a normal rate. The secondary mutations do
of viral integration. Genotyping is used to monitor the not affect the susceptibility of the virus to the drug, but
development of this antiretroviral drug resistance. Most rather help the virus replicate in the presence of the drug
of the antiretroviral drugs target the reverse transcriptase when one of its replication enzymes is not 100% func-
and protease enzymes, so these are the genes that are tional. Once a resistance genotype has been identified,
most often examined in genotyping procedures. the drug therapy of the patient should be changed as
To perform genotyping, viral RNA is extracted, and soon as possible to avoid the development of secondary
PCR is used to amplify the whole protease gene and mutations in the virus.
part of the reverse transcriptase gene. The products are The results of genotyping procedures are reported by
analyzed for the presence of mutations by sequencing, listing the mutations that have been identified in the pro-
hybridization onto high-density microarrays, or reverse tease and reverse transcriptase genes and the impact those
hybridization. Sequencing is performed most often, and mutations will have on each drug: no evidence of resis-
there are commercially available kits for Sanger sequenc- tance, possible resistance, resistance, or insufficient evi-
ing. Using these genotyping methods for resistance dence. The mutations are indicated by reporting a change
monitoring, however, is a high-cost and low-throughput in the amino acid that is coded by the changed codon,
method. Next-generation sequencing (NGS) along with where the wild-type amino acid is written, followed by
simpler sample collection and storage matrices (e.g., the position of the codon that is changed, followed by the
dried blood spots36) will facilitate and broaden resistance new amino acid. For example, a mutation in codon 184 of
monitoring as well.37 the reverse transcriptase gene from ATG to GTG results
To evaluate resistance, viral sequences are com- in an amino acid change from methionine to valine, or
pared with a reference sequence to identify the muta- M184V. This particular mutation makes the virus resis-
tions present. After the mutations have been identified, tant to the cytidine analog, lamivudine. Another muta-
their significance in terms of the impact on antiretroviral tion, Q151M, located at the dNTP-binding site of reverse
therapy is assessed, and this is generally accomplished transcriptase, confers resistance to reverse transcriptase
through computer algorithms. Resistance mutations inhibitors while maintaining sensitivity to lamivudine.
have been well characterized for individual agents, but As with all other molecular-based assays, HIV geno-
HIV-positive patients are more often on cocktails of typing procedures should employ adequate quality and
drugs rather than one drug. Therefore, the impact of a contamination controls. The sensitivity of the methods
mutation on multiple drugs is also considered. In addi- for detecting a minority of virions that contain muta-
tion, if a single virus has multiple mutations, the inter- tions in the midst of a majority of wild-type virions is
pretation of more than one mutation in the context of an important consideration. The sensitivity of the auto-
the others and with respect to multiple drugs becomes mated sequencing methods has been reported to be 20%,
more complex. Computer algorithms are used to analyze and NGS has a reported sensitivity of less than 1%.39
genotypes, taking into account primary and secondary Proficiency testing is provided by the CAP, and indepen-
mutations, cross-resistance, and the interactions between dent control materials for use in genotyping assays are
mutations that can affect resistance.38 commercially available.
320 Section III • Techniques in the Clinical Laboratory

Herpes viruses are frequent sources of human infec- infection is characterized by the presence of EBV-en-
tion. At least 25 viruses comprise the family Herpes- coded RNA (EBER). The virus can reactivate and is
viridae. Several herpes virus types frequently infect commonly found in the saliva of infected persons. EBV
humans, including herpes simplex virus (HSV), CMV, infection is associated with a variety of benign and
EBV, and varicella-zoster virus (VZV). In addition to malignant lesions, including Hodgkin disease, non-Hod-
causing overt disease, herpes viruses may remain silent gkin lymphoma, Burkitt lymphoma, gastric carcinoma,
and be reactivated many years after infection. and nasopharyngeal carcinoma. Although EBV is
HSV has a relatively large viral genome encoding present in these conditions, it is likely not the sole cause
at least 80 protein products. About half of these pro- of disease.
teins are involved in the interaction with the host cell Laboratory testing for EBV infection has been per-
or immune system. The other half control viral struc- formed by immunohistochemistry for EBV proteins
ture and replication. There are two types of HSV, HSV in tissue and testing serum for antibodies to EBV-
type 1 (HSV-1) and HSV type 2 (HSV-2). HSV-1 mainly associated antigens, including the viral capsid antigen,
causes cold sores or fever blisters; HSV-2 mainly causes the early antigen, and the EBV nuclear antigen (EBNA).
genital sores but can also cause mouth sores. HSV tests Confirmation may be done by differentiation of IgG and
are usually done for genital sores, although body fluids, IgM subclasses to the viral capsid antigen. EBER tran-
including blood, urine, tears, amniotic fluid, lavages, and scripts are primary targets for tissue analysis. Repeated
spinal fluid, may also be tested. or unique sequences in EBV DNA are targets of in situ
HSV was detected by viral culture, antigen, and anti- hybridization; however, the sensitivity of DNA probes
body tests before the application of PCR. Viral cultures is lower than those for EBER, due to the abundant pres-
sometimes had to be performed more than once because ence of the EBER transcripts.
the virus might not be detected at all times of infection. Southern blot analysis of EBV DNA, first described
The HSV antigen test was performed on a slide smear of in 1986,40 was based on variable numbers of terminal
material from the sore. Viral antigens are detected with repeat sequences at the ends of each EBV DNA mole-
labeled antibodies. The antibody test was performed on cule. Each infecting genome can contain up to 20 ter-
blood to detect antibodies to the viral antigens. Anti- minal repeat sequences. When EBV DNA is cut with
bodies may not be detectable 2 weeks to 3 months after BamH1, the resulting fragments vary in size, depending
initial infection; however, once the infection occurs, anti- on the number of repeat sequences. The fragments were
bodies remain in the person for life. Western blot tests visualized with a probe complementary to the variable
could distinguish HSV-1 and HSV-2 using type-specific repeat.
antigens to detect the corresponding type-specific anti- Amplification methods are now used for detect-
bodies. Type-specific testing is important for prognosis ing EBV in blood, body fluid, or tissue samples. EBV
and patient counseling. DNA can be amplified using primers complementary
PCR tests also offer the advantage of distinguishing to conserved EBV sequences, and strain typing can be
HSV-1 from HSV-2 directly without culturing. Methods achieved by amplification of polymorphic regions of
include standard PCR and quantitative PCR target- the viral genome. Quantitative PCR is used to deter-
ing type-specific HSV genes (Table 11.5). One of the mine EBV viral load in blood using a reference inter-
most common human viruses, EBV is a member of the nal control. A reported quantitative range of analysis is
herpes virus family. Most people become infected with 750 to 106 IU/mL.41 As with other such tests, the virus
EBV sometime during their lives. Children can become may still be present below the level of detection.
infected with EBV once they lose maternal antibody Another member of the herpes virus family is CMV.
protection, and most adults in the United States between Most people have been infected with CMV without
ages 35 and 40 have likely been infected with EBV. The obvious illness. Like other herpes viruses, CMV can
first infection during adolescence or young adulthood go dormant and reactivate. If symptoms occur, they are
causes infectious mononucleosis. mild, except in immunocompromised individuals. The
EBV establishes a permanent dormant infection in virus is shed in blood, urine, saliva, and other body
cells of the throat, blood, and immune system. Latent fluids but dies quickly outside of the host.
 Chapter 11 • Detection and Identification of Microorganisms 321

Molecular detection of CMV is performed on cell- stage. The development of chronic infections is due to
free plasma or other fluids. DNA is extracted and the antigenic envelope proteins that elicit the produc-
amplified by PCR using primers targeting the CMV tion of antibodies. These proteins are encoded by hyper-
polymerase (UL54) or glycoprotein B (gB) gene in variable regions much like antibody genes themselves,
regions that do not share sequence homology with other resulting in extensive variation in the envelope proteins
herpes viruses. Internal controls are included to avoid and the escape of the virus from antibodies.
false-negative interpretation due to PCR inhibition. The The initial approaches to HCV analysis are similar to
level of detection of standard laboratory assays is such those for HIV. Serology is used to detect the presence
that no PCR product will be produced from normal of antibodies against HCV. If the patient has HCV anti-
plasma, even from people previously exposed to CMV. bodies, the specificity of the antibodies for HCV anti-
Automated amplification and detection of CMV has an gens is measured by western blot, where the presence
analytical sensitivity as low as one viral copy per micro- of antibodies with multiple HCV specificities confirms
liter. Quantitative PCR can detect down to 40 genome the diagnosis.
copies of virus per microliter of blood. PCR assays A variety of nucleic acid amplification assays for
may be affected by the presence of mutations in the the qualitative detection and quantitation of HCV are
target genes that interfere with primer hybridization. available, including RT-PCR, transcription-mediated
These mutations may also confer resistance to antiviral amplification, and branched DNA. The qualitative HCV
therapy. RNA assays are performed on patients with positive
VZV (HHV3) is a herpes virus that infects younger HCV antibody results to confirm active infection or
and older humans. VZV is the causative agent of chick- on immunocompromised patients (who are often co-
enpox and shingles (also called herpes zoster). The virus infected with HIV) in whom HCV infection is suspected
has a large genome containing over 70 open reading but antibody tests are negative. The quantitative HCV
frames (ORFs). Polymorphisms in the ORF are used to RNA assays are used as for HIV to determine the viral
determine the strain variations and genotype of viruses load and to monitor viral replication in response to anti-
from different geographical areas. An attenuated live viral therapy. The viral load and the HCV genotype are
virus, VZV Oka, developed by selection in cell cul- used to determine the therapeutic protocol, both type of
tures, is the parental strain source of a VZV vaccine, drug(s) as well as duration.
which contains a mixture of genotypically distinct viral Six genotypes (1a/1b, 2, 3, 4, 5, and 6) and more than
variants. 50 subtypes of HCV have been identified. The geno-
VZV is neurotropic, remaining latent in nerve cells type of HCV present in a given patient determines the
and possibly reactivating years after primary infections treatment protocol used on that patient, as particular
to result in shingles, or palsy. It is found in more than genotypes are associated with certain antiviral resistance
90% of young people living in temperate climates. There patterns. The HCV genotype is determined by analyzing
is no animal reservoir for VZV. Clinical tests include the core and/or 5′ untranslated regions of the genome.
enzyme-linked immunosorbent assays and PCR methods Laboratory methods available for HCV genotyping are
for detection of antibodies to VZV in serum. Qualitative PCR with restriction fragment length polymorphism
PCR methods to detect VZV include PCR with hybrid- (RFLP) analysis and reverse hybridization and direct
ization or direct gel electrophoresis. Quantitative PCR DNA sequencing. A PCR with melt-curve method has
kits include internal controls to detect amplification also been described.42
inhibition. It has been observed that patients who have a 2 log10
HCV is an enveloped, single-stranded RNA virus of decrease in HCV RNA 12 weeks after treatment begins
the Flaviviridae viral family. HCV causes viral hepatitis have a 65% chance of responding, defined by the lack
and cirrhosis and is also associated with causing hepato- of detection of HCV RNA in qualitative assays where
cellular carcinoma. The virus is transmitted parenterally the detection limit is 50 to 100 copies of virus/mL of
like HIV. Acute infections are often asymptomatic and plasma. Patients who do not have a 2 log10 decrease in
are rarely associated with jaundice; thus, patients with HCV RNA 12 weeks after treatment begins have less
acute HCV infections are usually not detected at this chance of responding. Determining the genotype of the
322 Section III • Techniques in the Clinical Laboratory

virus is also critical to predicting treatment outcomes; percentage of HPV DNA infected cells that are pre-
for example, genotypes 2 and 3 will respond better to cancerous. The PCR genotyping detects 37 high- and
particular treatments than genotype 1. low-risk HPV types by hybridization of PCR products
HPV is a double-stranded DNA virus recognized as to immobilized probes for each of the mutations. Direct
oncogenic in several human cancers. There are over sequencing may also be used to detect more HPV types.
200 HPV types based on sequences of the viral genome Methods may differ, not only in the sensitivity to
compared with known HPV genomes. Five evolution- target viruses but with the ability to detect concurrent
ary HPV genotype groups (α, β, γ, μ, and ν) have been infection with different HPV types. In all cases, patient
defined. The largest, the α group, contains 64 types that management decisions reflect patients’ overall cytology
mainly infect mucosal epithelia in the anogenital tract history and other risk factors in addition to the presence
and include the high risk (HR) types (HPVs 16, 18, or absence of high-risk HPV types.
31, 33, 35, 39, 45, 51, 56, 58, 59, 68, 73, 82) that have Respiratory viral infections are a major cause of hos-
been classified as oncogenic and are found to cause ano- pitalizations in young children and elderly people in
genital cancers. The next largest group is the β-group the United States. Over a dozen respiratory pathogens
HPVs that contains more than 50 characterized types (viral and bacterial) are commonly encountered in the
that mainly infect cutaneous epithelia. The β group and medical laboratory. Treatment and control of the spread
ultraviolet (UV) radiation exposure are associated with of infection will depend on which of these are infecting
nonmelanoma squamous cell carcinomas, a common a patient. For viral infections, molecular methods have
human cancer. HPVs of the remaining three groups (γ, been designed to detect multiple species in single anal-
μ, and ν) normally cause only benign disease.43 HPV yses. For example, a real-time PCR method can detect
HR types are responsible for over 95% of cervical squa- influenza A, influenza B, and RSV. Influenza A subtyp-
mous carcinoma. The presence of high-risk HPV DNA ing by melt-curve analysis has been used as a screen-
in conjunction with an equivocal or ambiguous cytology ing method to detect the 2009 influenza A (H1N1) virus
result (atypical squamous cells of unknown significance from nasal swabs.
[ASC-US]) indicates an increased risk for having an
underlying cervical neoplasm. Persistent infection with
high-risk HPV may be the main risk factor for the devel- Advanced Concepts
opment of high-grade cervical neoplasia and cancer.
Women with normal cervical cytology who are negative Amplification methods that target the HPV
for the high-risk HPV types are at low risk for having or L1 gene may not detect virus that has integrated
developing cervical precancerous lesions. into the host DNA because that gene region may
It is difficult to culture HPV in the laboratory, so be lost, causing false-negative results. Coin-
detection relies on molecular testing. Two approaches to fection with two or more HPV types will lower
molecular detection of HPV are hybridization methods overall PCR efficiency for each type or selectively
and amplification methods. The latter methods include amplify only one type, also resulting in false-
target amplification, such as PCR, and signal amplifi- negative results.
cation, such as hybrid capture. Hybridization tests can
detect a minimum of 4,000 to 5,000 viral genomes.
These tests also have the capacity for subtyping. HPV RSV is detected using several assays. An ASR for RSV
expresses its early genes, E1 through E8, shortly after A + B RNA uses NASBA technology. A bead array
host infection. The E6 and E7 genes are overexpressed technology can simultaneously detect RSV A and B,
after integration of oncogenic genotypes of the HPV influenza A, nonspecific influenza A, H1, H3, influ-
genome in the host genome. E6 and E7 gene products enza B, parainfluenza 1, parainfluenza 2, parainfluenza
are involved in the cellular transformation to cervical 3, metapneumovirus, rhinovirus, and adenovirus. The
cancer. Measurement of E6 and E7 mRNA expression test includes an MS-2 bacteriophage internal control
in specific cell types identified by flow cytometry is a and a lambda bacteriophage positive control. It aided
specific indication of cellular transformation in the small in the detection of 2009 influenza A/HIN1 (swine flu)
 Chapter 11 • Detection and Identification of Microorganisms 323

but could not identify the hemagglutinin gene of the

Mycology
2009 influenza A/H1N1 directly.44 Seasonal and H1N1
flu viruses develop resistance to antiviral agents through Fungi are among the most ubiquitous microorganisms
sequence alterations.45 These mutations can be detected causing clinical infection. Traditional methods of iden-
by direct sequencing. Digital PCR methods have also tification by phenotype have become more difficult
been developed to detect the frequently occurring alter- with the expanding diversity of organisms. Molecular
ation H275Y in the neuraminidase gene (H274Y in methods, particularly sequencing and PCR, have allowed
N2 nomenclature).46 for the detection and typing of fungi with greater sensi-
BK and JC viruses are human polyomaviruses, the tivity, specificity, and speed.
primate counterpart of which is SV40. BK, JC, and Fungi are important causes of human disease, espe-
SV40 share sequence and antigenic homology. They are cially in immunocompromised patients. In addition,
double-stranded DNA viruses with 5,000-bp genomes laboratory-acquired infections from fungi are a major
encoding 5 transcripts. Most polyomavirus infections are risk for laboratory personnel. Fungal infections are most
subclinical in adults. Infected children develop respira- often diagnosed by direct staining methods and isolation
tory symptoms, and some have cystitis. Several reagent of the causative agent in culture. As for other organisms,
sets are available for detection of BK or JC viruses in the traditional smears and cultures are affected by sensitiv-
clinical laboratory, including quantitative PCR tests.47 ity, organism viability, and the length of time required
for the organism to grow. Despite these problems, direct
smears and isolation of fungi are still the major methods
Mass Spectrometry
for detecting fungi in clinical samples.
Application of mass spectrometry to bacteriology has Fungi growing in culture are typically identified by
been extended to virology to a limited degree. Viruses their microscopic and macroscopic morphologies or
have low concentrations of high-molecular-weight pro- by using fluorescent antibodies. For some fungi, such
teins that are more difficult to assess by peptide pro- as Histoplasma, Blastomyces, Coccidioides, and Cryp-
files. Confounding viral detection is cell debris from the tococcus neoformans, gene probes have been devel-
cultures in which the viruses were cultivated. MALDI oped to confirm the identity of the organism growing
analysis of PCR-amplified viral nucleic acid has been in culture.51 Using DNA probes for these organisms is
successfully applied to clinical detection of HSV, HCV, faster and less hazardous than determining the micro-
HPV, enteroviruses, and respiratory viruses.48 Multiplex scopic morphology.
PCR with MALDI (PCR-mass assay) has been shown to Broad-range PCR and subsequent analysis are also
identify multiple viruses or multiple viral subtypes in a used for clinical analysis of fungi. In these approaches,
single test.49 primers anneal to DNA sequences that are common to
PCR-based genotyping of frequently rearranged most of the clinically relevant fungi, such as Candida,
viruses such as influenza where recombinant viruses Aspergillus, Rhizopus and other Zygomycetes, and
arise from coinfection with distinct viral stains has Histoplasma and other dimorphic fungi. Once the
proven more difficult. PCR amplification in these cases sequences are amplified, hybridization to species-specific
becomes problematic as primer-binding sights are lost probes or sequencing is used to identify the fungus to the
or changed in the recombinant strains. These reassorted genus or genus and species level. In-house-developed
flu virus strains are also difficult to detect by immuno- real-time PCR methods are also used for direct iden-
logical methods as the antigenic determinants become tification of Aspergillus and Pneumocystis carinii.
altered. Characterization of the type of virus is impor- Blastomycetes, Coccidioides immitis, and Histoplasma
tant for early intervention and prevention of infectious capsulatum are detected with direct probe hybridiza-
spread. For this application, mass spectrometry has been tion assays. The probe methods can also be multiplexed.
shown to successfully genotype viruses using whole- PFGE is used in laboratory-developed methods for
virus protein digests.50 Human influenza and parainflu- molecular typing of yeasts.
enza viruses can be subtyped and lineages traced by this Molds can be typed by PCR and sequencing of ITS
method. regions or 28S rRNA. ITS sequences of DNA extracted
324 Section III • Techniques in the Clinical Laboratory

from 24 to 48 mold cultures are compared with those of Recognition of these issues has made the develop-
reference strains. This method is used in industrial and ment of molecular-based assays for parasite detection
environmental settings to positively identify Paecilo- and identification more practical. Nucleic acid isolation
myces species, which are saprophytic filamentous fungi for molecular testing has been addressed by using several
found in soil and as air and water contaminants. Paeci- methods, including extraction and extraction-free,
lomyces lilacinus and Paecilomyces variotii are increas- filter-based protocols for preparation of DNA templates
ing causes of opportunistic human infections generally from oocysts and microsporidia spores. PCR methods
associated with the use of immunosuppression therapy, are modified to accommodate sequence content, for
saline breast implants, or ocular surgery. Although these example, low GC content in malaria.
two species present differences in their in vitro suscepti- PCR assays have been developed for the detection of
bility to antifungal agents, requiring identification before Trypanosomes, Plasmodia, Toxoplasma, Entamoebae,
treatment, molecular testing in the clinical laboratory is and Cryptosporidium in hosts and water sources. Multi-
not well developed. plex qPCR methods can also measure infection rates and
MALDI-TOF application to detection of fungi has organism loads in hosts and nonhuman vectors (carriers
been limited by their biological complexity and different of infection).54
fungal growth phases. Furthermore, their thick cell walls In-house-developed real-time PCR methods are used
require chemical and physical disruption methods. Cell in clinical laboratories for identification of Babesia,
walls are extracted with trifluoroacetic acid, or formic Trichomonas microti in blood and from ticks, Enceph-
acid and acetonitrile, possibly followed by physical dis- alitozoon species microsporidia, and Trichomonas vag-
ruption with beads. inalis. Multiplex PCR methods have been designed to
Mass spectrometry methods for identification of S. simultaneously detect multiple parasites, for example,
cerevisiae were first developed in 2001.52 Reproducible multiplex real-time PCR assays for detection of Entam-
spectra have since been observed for other yeasts includ- oeba histolytica, Giardia lamblia, and Cryptosporid-
ing Candida and Cryptococcus. MALDI-TOF may also ium parvum simultaneously or E. histolytica, Giardia
offer a reliable, rapid approach to testing for fungal intestinalis, and Cryptosporidium spp. simultaneously
sepsis.53 in stool samples. The multiplex PCR methods also
include an internal control to determine the efficiency
of the PCR and detect inhibition in the sample, which
Parasites
is likely in stool samples. The development of mul-
Parasites are typically detected and identified by mor- tiplex PCR assays to detect multiple parasites in stool
phology directly in clinical specimens, a method of samples is extremely useful. First, multiple parasites
diagnosis subject to false-negative results in cases of can cause diarrhea, and morphology is the only way to
low organism concentrations and depending greatly on differentiate between causative agents. Second, patients
appropriately trained personnel. Molecular-based testing can have multiple intestinal parasites at the same time,
for parasites has been limited mainly because parasites and laboratory detection of the presence of all parasites
are not a major cause of disease in developed coun- is important. Finally, multiple parasites are transmitted
tries. Increasingly, however, travelers from parasite- through the same source; thus, detection of all parasites
endemic countries bring parasites to developed countries and appropriate control measures will reduce large-scale
and serve as a reservoir for transmission. In the labo- outbreaks.
ratory, nucleic acid template preparation from oocysts
and spores of protozoan parasites is complicated by the
nature of the organism and its resistance to disruption ANTIMICROBIAL AGENTS
and lysis. Parasites found in complex matrices such as
stool samples present a further difficulty due to inhi- Antimicrobial agents are of two types, those that
bition of PCR and other enzymatic assays. However, inhibit microbial growth (-static, e.g., bacteriostatic,
expertise in identifying parasites by morphology is fungistatic) and those that kill organisms outright
declining, demanding alternative methods of surveil- (-cidal, e.g., bacteriocidal, fungicidal). Antimicrobial
lance and detection of these organisms. agents for use in clinical applications are designed to be
 Chapter 11 • Detection and Identification of Microorganisms 325

Essential
TABLE 11.8 Mode of Action metabolism
of Antimicrobial Agents Cell wall integrity
Protein
synthesis
Mode of Action Examples

Disrupts cell wall synthesis Beta-lactams (penicillins and

or integrity cephalosporins)
Glycopeptides (vancomycin)

Disrupts cell membrane Polymyxins (polymyxin B)

structure or function Bacitracin Membrane
integrity
Inhibits protein synthesis Aminoglycosides (gentamicin)
Tetracyclines
Macrolides (erythromycin)
Lincosamides (clindamycin)
Nucleic acid
Inhibits nucleic acid Quinolones (ciprofloxacin) metabolism
synthesis or integrity Metronidazole FIGURE 11.5 Sites of antimicrobial action. Depending on
Inhibits metabolite Sulfamethoxazole the type of organism, several structures can be affected by anti-
synthesis Trimethoprim microbial agents. All of these are essential for cell growth and
survival.

selective for the target organism with minimal effect on antibiotics such as vancomycin can lead to the devel-
mammalian cells. These agents are also intended to dis- opment of resistant clones of organisms. These clones
tribute well in the host and remain active for as long as may persist in low numbers below the detection levels
possible (long half-life). Ideally, the agents should have of routine laboratory sensitivity testing methods.
-cidal (rather than -static) activity against a broad spec-
trum of microorganisms.
Another way to classify antimicrobial agents is by Advanced Concepts
their mode of action (Table 11.8). The ultimate effect of
these agents is to inhibit essential functions in the target The first antibiotics isolated were natural secretions
organism (Fig. 11.5). A third way to group antimicrobial from fungi and other organisms. Synthetic mod-
agents is by their chemical structure. For example, there ifications of these natural agents were designed
are two major types of agents that inhibit cell wall syn- to increase the spectrum of activity (ability to
thesis, the β-lactams with substituted ring structures and kill more organisms) and to overcome resistance.
the glycopeptides. For example, cephalosporins include such first-
generation agents as cephalothin and cefazolin that
are active against Staphylococcus, Streptococcus,
Resistance to Antimicrobial Agents
and some Enterobacteriaceae. A second generation
Microorganisms naturally develop defenses to antimi- of cephalosporins—cefamandole, cefoxitin, and
crobial agents. At the molecular level, resistance can cefuroxime—is active against more Enterobacteria-
arise from alerted target binding, active extrusion, or ceae and organisms resistant to β-lactam antibiotics.
inaccessibility of the drug or microbial enzymes that A third generation—cefotaxime, ceftriaxone, and
inactivate the drug. Multidrug-resistant organisms may ceftazidime—is active against P. aeruginosa as
have one or more of these characteristics. A single- well as many Enterobacteriaceae and organisms
nucleotide variant in a drug target or transport protein resistant to β-lactam antibiotics. The fourth gener-
can result in resistance. Resistant Staphylococcus, Pseu- ation, cefepime, is active against an extended spec-
domonas, and Klebsiella spp. are becoming common- trum of organisms resistant to β-lactam antibiotics.
place in health-care institutions. Long-term therapy with
326 Section III • Techniques in the Clinical Laboratory

TABLE 11.9 Resistance Mechanisms TABLE 11.10 Genes Conferring Resistance

to Antimicrobial Agents in Particular Organisms
Examples of
Mechanism Example Agents Affected Antimicrobial Gene(s) Conferring
Organism Agent Resistance
Destruction/ β-lactamases β-lactams
modification of agent aminoglycosides Staphylococcus Oxacillin mecA
aureus
Elimination of agent Multidrug β-lactams,
efflux systems fluoroquinolones, Streptococcus Penicillin pbp1a and pbp1b
macrolides, pneumoniae
chloramphenicol,
trimethoprim Gram- β-lactams tem, shv, oxa, ctx-m
negatives
Altered cell wall Thick cell Vancomycin
structure walls that β-lactams Enterococcus Vancomycin vanA, vanB, vanC,
exclude agent vanD, vanE, vanG
Altered agent
binding sites Salmonella Quinolones gyrA, gyrB, parC, parE

Alternate metabolic Altered Sulfonamides, Mycobacterium Isoniazid katG, inhA

pathways enzymes trimethoprim tuberculosis

Rifampin rpoB

There are several ways in which microorganisms

develop resistance (Table 11.9). First, bacteria can
produce enzymes that inactivate the agent. Examples will eventually take the place of those without the muta-
of this resistance mechanism are seen in S. aureus and tion, which are less able to survive and procreate. This
N. gonorrhoeae that produce β-lactamase, an enzyme process is stimulated by antibiotic exposure, especially
that cleaves the β-lactam ring of the β-lactam antimicro- if the levels of antibiotics are less than optimal. For
bials such as the penicillins. Cleavage of the β-lactam example, S. aureus developed resistance to antibiot-
ring destroys the activity of penicillin, rendering the ics that target its penicillin-binding protein (PBP1) by
organism resistant to its antimicrobial action. Second, replacing PBP1 with PBP2a encoded by the mecA gene.
organisms produce altered targets for the antimicrobial PBP2a found in methicillin-resistant S. aureus (MRSA)
agent. Mutations in the gene encoding a penicillin- has a low binding affinity for methicillin.
binding protein—for example, a change in the struc- Another genetic resistance mechanism is the acqui-
ture of the protein—render penicillin unable to bind to sition of genetic factors from other resistant organisms
its target. Finally, bacteria exhibit changes in the trans- through transformation with plasmids carrying resis-
port of the antimicrobial agent either into or out of the tance genes or transduction with viruses carrying resis-
cell. An example of this mechanism is seen in gram- tance genes. Genetic factors can also be transferred
negative bacteria that change their outer membrane from one bacterium to another by conjugation. Genet-
proteins (porins) in order to decrease the influx of the ically directed resistance can pass between organisms
antimicrobial agent. If the agent cannot get into the cell of different species. For example, MRSA (vancomycin-
and bind to its target, then it is not effective in inhibiting sensitive S. aureus) can gain vancomycin resistance
or killing the bacterium. from vancomycin-resistant Enterococcus faecalis. Van-
All these resistance mechanisms involve a genetic comycin and other glycopeptides act by preventing the
change in the microorganism (Table 11.10). These cross-linking of the peptidoglycan, thereby inhibiting
genetic changes are most commonly brought about by cell wall production. Several genes have been found in
mutation and selection processes. If a mutation results in enterococci that encode altered binding proteins: vanA,
a survival or growth advantage, cells with the mutation vanB, vanC, vanD, vanE, and vanG. The expression of
 Chapter 11 • Detection and Identification of Microorganisms 327

ORF 1 ORF 2 vanA vanS vanH vanA vanX vanY vanZ

Tn1546

58K VRSA
plasmid

FIGURE 11.6 Vancomycin-resistant S. aureus (VRSA) plasmid carrying transposon Tn1546 with vancomycin resistance genes.

vanA and vanB is inducible and transferred from cell to resistance for a given organism and antimicrobial agent
cell by plasmids carrying vancomycin resistance genes pair. The determination of MICs to detect antimicro-
on a transposon55 (Fig. 11.6). The resulting vancomycin- bial resistance is a phenotypic method. Although MIC
resistant S. aureus (VRSA) uses lactic acid instead of methods are well established, and the results are gener-
alanine to build its cell wall. The VRSA cell wall, then, ally reliable with regard to in vivo effectiveness of an
does not contain the target structure (D-ala-D-ala) for agent for an organism, the results are sometimes diffi-
vancomycin. Development of resistance to particular cult to interpret and the procedures are time-consuming,
drugs occurs with particular mutations: rpoB mutation taking at least 48 hours after the specimen is collected.
is associated with rifampin resistance, and mutations in Molecular methods that detect genes directly involved
katG, inhA, ahpC, and ndh genes are associated with in the resistance of an organism to a particular agent offer
resistance to isoniazid.56 a more straightforward prediction of antibiotic resis-
tance. There are four reasons for using molecular-based
Molecular Detection of Resistance methodologies. First, when an organism has an MIC at
or near the breakpoint of resistance, detection of mutated
Susceptibility to antimicrobial agents is determined by
genes contributing to resistance would be irrefutable
phenotypic or genotypic methods. The phenotypic devel-
evidence of the potential ineffectiveness of the agent.
opment of resistance is detected by performing in vitro
Second, genes involved in the resistance of organisms to
susceptibility testing. Testing for altered sensitivity to
antimicrobial agents can be detected directly in the clin-
antimicrobial agents is of clinical significance especially
ical specimen closer to the time of collection and save
when organisms persist in patients being treated with
the time required to isolate the organism and perform
antimicrobial agents that are generally considered effec-
phenotypic MIC determinations on isolated colonies.
tive against the particular isolate or when large numbers
With no requirement for culturing potentially dangerous
of organisms are observed in normally sterile fluids,
microorganisms, there is less chance of hazardous expo-
such as blood, cerebrospinal fluid, or urine. Phenotypic
sure for the technologist as well. Third, monitoring the
methods are generally used for aerobic bacteria, some
spread of a resistance gene in multiple isolates of the
mycobacteria, and yeast. For other organisms, such as
same organism is more useful in epidemiological inves-
viruses and filamentous fungi, phenotypic methods
tigations than following the trend in the MIC. Finally,
are not well standardized. Phenotypic methodologies
molecular methods are considered the gold standard for
include disk diffusion, broth dilution, and direct detec-
validation of new phenotypic assays.
tion of resistance factors such as β-lactamase.
Susceptibility testing measures the minimum inhib-
Beta-Lactam Antibiotic Resistance
itory concentration (MIC) of an antimicrobial agent,
or the least amount of antimicrobial agent that inhibits The earliest commercialized antibiotic, penicillin, was
the growth of an organism. Established guidelines define first used therapeutically in the early 1940s (Fig. 11.7).
the MICs interpreted as indications of susceptibility or Soon after that, resistance to penicillin through the
328 Section III • Techniques in the Clinical Laboratory

R O
O ONa
CH3 O
C O H
NH S N CH3
CH3 O
HN S CH3
O O N CH3 N
H S CH3 CH3 O
HC C C H
O
CH3 HO N CH3
C N CH O
–
COO
O FIGURE 11.9 Penicillin substitutes include methicillin (left)
FIGURE 11.7 Structure of penicillin, showing the carbon- and oxacillin (right).
nitrogen (CCCN) beta-lactam ring.
community. As described previously, expression of an
+
D + altered penicillin-binding protein (PBP2’ or PBP 2a
encoded by the mecA gene) is the resistance mechanism.
D N O NH
S
␤-Lactamase The antibiotics cannot bind to the altered target and
O N A –O2C
S therefore have no effect on the bacterial cells.
O OH– Rapid identification of MRSA isolates in clinical spec-
N A
CO2– imens by direct detection of mecA is critical for effective
H
CO2– patient management and prevention of infections occur-
ring in hospitals due to MRSA. PCR and other amplifi-
D cation assays have been developed for direct testing of
O NH clinical samples, and many assays have been tested for
+ sensitivity and specificity.
S A–
–O2C The stereochemical structure of the carbapenems
N makes them more resistant to inactivation by most
CO2–
plasmid and chromosomal-mediated β-lactamases than
the penicillins. After widespread use of these agents,
FIGURE 11.8 The reaction catalyzed by beta-lactamase however, resistance developed primarily through the
results in cleavage of the beta-lactam ring through an interme- production of carbapenem-hydrolyzing β-lactamases
diate product (bracketed). (carbapenemases).58
MALDI-TOF mass spectrometry peptide profiles
production of β-lactamases (Fig. 11.8) was recorded.
can discriminate resistant strains of organisms such as
Streptococcus pyogenes is one of the very few organ-
MRSA and VRE. In a mass spectrometry β-lactamase
isms that are still predictably susceptible to penicillin.
assay, the antibiotic is mixed with the bacterial culture.
Modified β-lactam molecules including the cephalo-
After incubation, the bacteria are removed by centrifuga-
sporins and carbapenems were subsequently developed.
tion, and the supernatant is analyzed for the hydrolyzed
Penicillin and the other β-lactam antimicrobials inhibit
form of β-lactam. This method can detect resistance to
bacteria by interfering with an enzyme that is involved
penicillin and a variety of β-lactam antibiotics, includ-
in the synthesis of the bacterial cell wall.
ing ampicillin, piperacillin, ceftazidime, imipenem, and
To counter the production of the bacterial
others.59 Modifications of the method have been aimed
β-lactamases (also known as penicillinases), penicillinase-
at increased speed and accuracy. Carbapenemase pro-
resistant penicillins—for example, methicillin or oxacillin
duction from anaerobic bacteria in less than 3 hours has
(Fig. 11.9)—were designed. Staphylococcal infections
been reported.60,61
were treated successfully with methicillin/oxacillin until
the emergence of resistance to these agents was first
Glycopeptide Antibiotic Resistance
observed in 1965.57 MRSA and methicillin-resistant
coagulase-negative staphylococci became a major cause Glycopeptide antibiotics were originally isolated from
of infections acquired in the hospital as well as in the plant and soil bacteria. Their structures contain either
 Chapter 11 • Detection and Identification of Microorganisms 329

HO The specificity of PCR and multiplex PCR was high

NH2
OH compared with the isolation of VRE in culture. Quan-
O
OH titative PCR has also been used to detect VRE in fecal
O surveillance specimens. Because PCR is faster than tra-
OH ditional culture methods and has comparable sensitiv-
OO ity and specificity to culture, it is an attractive method
Cl
O O for screening large numbers of samples for a particular
HO
6
Cl
4
OH
target.
O O
H H
O N N
N N NH Antimicrobial Resistance in M. tuberculosis
H H H
HN O O O N
O The use of molecular methods greatly improved antimi-
O– NH2
crobial resistance detection in M. tuberculosis because
OH
O OH traditional methods of determining antimicrobial suscep-
HO tibility took days, if not weeks, for this organism. The
FIGURE 11.10 Structure of vancomycin. longer a patient with tuberculosis (TB) is inadequately
treated, the more likely the development of resistance
and possible spread of the resistant organisms. Evolution
a glycosylated cyclic or polycyclic peptide. These anti- of drug resistance of M. tuberculosis in a noncompliant
biotics inhibit peptidoglycan synthesis in the cell wall patient for over 12 years was reported where subpopu-
of susceptible organisms (principally gram-positive lations of the organism emerged due to the acquisition
cocci). Glycopeptide antibiotics include vancomycin and accumulation of gene mutations that rendered the
(Fig. 11.10), teicoplanin, and ramoplanin; oritavancin, organism resistant to isoniazid, rifampin, and strepto-
dalbavancin, and telavancin are semisynthetic glyco- mycin.64 Many nucleic acid amplification protocols have
peptide antibiotics. Enterococcus was the first organism been developed to directly detect mutations in the genes
in which glycopeptide resistance was reported.62 Since associated with resistance to isoniazid and rifampin. In
then, vancomycin resistance has been observed in other general, these assays have demonstrated excellent sensi-
organisms. tivity and specificity and provide rapid determination of
The mechanism of vancomycin resistance can be drug susceptibility either directly from sputum or from
acquired or intrinsic to the organism. The antibiotic cultures.
binds and weakens cell wall structures of susceptible Thus, the advantages of using nucleic acid amplifi-
bacteria, ultimately causing cell lysis. Either the pres- cation assays for the determination of drug resistance
ence of the antibiotic itself or the weakening of the cell include the rapid and specific detection of mutations in
wall activates a set of van genes whose products modify genes associated with resistance to particular antimicro-
the cell wall structure such that the antibiotic no longer bial agents that provides irrefutable evidence of resis-
binds. The van genes are carried on a cassette localized tance in a short period.
to a plasmid or on the bacterial chromosome. Resistance
arises from enzymatic modifications of the aminogly-
cosides by acetyl-, adeno-, and phospho-transferases. MOLECULAR EPIDEMIOLOGY
A universal database for the detection of the modified
drugs by MALDI-TOF MS has been established. To An epidemic is a disease or condition that affects many
date, these methods are not yet in routine laboratory use unrelated individuals at the same time; a rapidly spread-
because they have not achieved the ease and reproduc- ing outbreak of an infectious disease is an epidemic.
ibility of routine diagnostic systems.63 A pandemic is a disease that sweeps across wide geo-
PCR was used to detect the vanA, vanB, vanC1, graphical areas. Epidemiology includes collection and
and vanC2 resistance genes in fecal samples as a way analysis of environmental, microbiological, and clinical
to screen for vancomycin-resistant enterococci (VRE). data. In microbiology, studies are performed to follow
330 Section III • Techniques in the Clinical Laboratory

the spread of pathogenic organisms within a hospital sequencing methods. Mass spectrometry databases
(nosocomial infections), from the actions of a physician continue to accumulate for detailed typing of bac-
(iatrogenic infections), and in a community. Molecular teria and fungi by peptide profiles. The ability to
epidemiology is the study of causative genetic and envi- discern differences with increasing detail enhances the
ronmental factors at the molecular level to ascertain the capability to type organisms regardless of their com-
origin, distribution, and best strategies for prevention of plexity. All methods, however, have benefits and lim-
disease. In infectious disease, these efforts are facilitated itations with regard to instrumentation, methodology,
by the ability to determine the genetic similarities and and interpretation.
differences among microbiological isolates. Molecular methods are based on DNA and amino
In the laboratory, molecular methods are very useful acid sequences. Nucleotide and amino acid sequences
for identifying and typing infectious agents. In a single range from highly conserved across species and genera
patient, this is informative for therapeutic efficacy; in to unique to each organism. Some of these are strain-
groups of patients, it provides information for infec- or species-specific sequences but are still used for epi-
tion control. Typing systems are based on the premise demiological testing because these methods are highly
that clonally related isolates share molecular charac- reproducible and, depending on the targets, can dis-
teristics distinct from unrelated isolates. Molecular criminate between even closely related organisms. Most
technology provides analytical alternatives from the (but not all) molecular methods offer definitive results
chromosomal to the nucleotide sequence level. These in the form of DNA sequences, peptide profiles, or gel
genotypic methods, in addition to established pheno- band and peak patterns that can be interpreted objec-
typic methods, enhance the capability to identify and tively, which is less difficult than phenotypic determi-
type microorganisms. Whereas phenotypic methods are nations that often require experienced judgment.65 With
based on a range of biological characteristics, such as commercial systems, the performance has become rel-
antigenic type or growth requirements, genotypic pro- atively simple for some molecular epidemiology tests,
cedures target genomic or plasmid DNA (Table 11.11). whereas others require a higher level of laboratory
Genome scanning methods, such as restriction enzyme expertise.
analysis followed by PFGE, are used to find genetic
similarities and differences, as are amplification and
Molecular Strain Typing Methods
for Epidemiological Studies
In community or clinical settings, the same organism
TABLE 11.11 Epidemiological Typing Methods might be isolated multiple times, whether in the same
patient or from different patients. The physician has to
Class Methods determine whether collected isolates were independently
acquired, that is, came from different sources, or if they
Phenotypic Biotyping, growth on selective media came from the same source. With this knowledge, the
Antimicrobial susceptibility
Serotyping, immunoblotting actions are taken to control the transmission of the organ-
Bacteriophage typing ism, especially if it is being transmitted from a common
Protein, enzyme typing by electrophoresis source and that source has been identified. Most of the
MALDI-TOF mass spectrometry time, these analyses are performed on organisms that
Genotypic Plasmid analysis
have been transmitted nosocomially, but sometimes pro-
Restriction endonuclease mapping cedures to determine relatedness are performed on iso-
Pulsed-field gel electrophoresis lates from community outbreak situations.66
Ribotyping Many laboratory methods can be used to determine
Arbitrarily primed PCR, RAPD PCR the relatedness of multiple isolates, both phenotypic
Melt-curve analysis
REP-PCR, ERIC PCR, ITS, spa typing
(e.g., by MALDI-TOF, serology, and antimicrobial sus-
Mass parallel sequencing ceptibility patterns) and genotypic (e.g., PFGE and ribo-
typing). The phenotypic methods suffer from a lack of
 Chapter 11 • Detection and Identification of Microorganisms 331

reproducibility and their ability to discriminate between (0)

isolates. Even after recent advances in mass spectrom- 1 2 3 4 5 6 7
etry, genotypic methods are used almost exclusively to
type bacterial strains to determine the relatedness of
multiple isolates.

Plasmid Analysis
Plasmid analysis involves isolation and restriction
mapping of bacterial plasmids. The same bacterial strain
can have different plasmids carrying different pheno- 1 2 3 4 5 6 7
types or resistance patterns. For this analysis, plasmid
DNA is isolated from the specimen or culture and then
digested with restriction enzymes. Plasmids are distin-
guished by gel electrophoresis patterns of fragments
generated when cut with the appropriate enzymes.
Restriction analysis can also be performed on chro-
mosomal DNA of organisms with small genomes. For
organisms with larger genomes, whole genome analysis
with restriction enzymes that cut frequently enough to
identify plasmids will yield complex patterns that are
more difficult to interpret.

Pulsed-Field Gel Electrophoresis

Most molecular epidemiological tests are performed
using PFGE, which can identify organisms with larger
genomes or multiple chromosomes. For PFGE analysis,
the DNA is digested with restriction enzymes that cut
infrequently within the genomic sequences. The result-
ing large fragments (hundreds of thousands of base
pairs) are resolved by PFGE. Banding patterns will
differ depending on the chromosomal DNA sequence
FIGURE 11.11 PFGE of coagulase-negative Staphylococcus
of the organisms (Fig. 11.11). Tenover and colleagues
showing the outbreak (O) and four test strains (top panel). The
devised a system to interpret the banding pattern of a gel image is shown in the bottom panel. The fragment shifts
test organism compared to that of the strain of an iden- are marked in the top panel. Strains in lanes 4, 5, and 6 are the
tified or reference organism.67 The interpretation of same but different from the outbreak strain (lane 2). The strain
PFGE results follows the “rule of three” (Table 11.12), a in lane 3 is the same as the outbreak strain. The strains in lanes
method that has been used for typing numerous species, 4, 5, and 6 have at least five genetic differences and are not
including strains of Pseudomonas aeruginosa, Myco- related to the outbreak. Lanes 1 and 7, molecular-weight
bacterium avium, Escherichia coli, N. gonorrhoeae, markers. (Courtesy of Mary Hayden, MD, Rush Medical Laborato-
VRE, and MRSA. Intralaboratory and interlaboratory ries, Rush University Medical Center, Chicago, IL.)
computerized databases of band patterns can be stored
for reference. A national PFGE database is stored at
Restriction Fragment Length
the CDC (www.cdc.gov/pulsenet). One disadvantage
Polymorphism Analysis
of using PFGE to type strains is the time involved to
perform the assay; it can take 2 to 3 days to complete RFLP analysis by Southern blot is the same technique
one analysis. first used to identify and investigate human genes. This
332 Section III • Techniques in the Clinical Laboratory

TABLE 11.12 Criteria for PFGE Pattern Interpretation

Category Genetic Differences* Fragment Differences* Epidemiological Interpretation

Indistinguishable 0 0 Test isolate is the same strain as the outbreak strain

Closely related 1 2–3 Test isolate is closely related to the outbreak strain

Possibly related 2 4–6 Test isolate is possibly related to the outbreak strain

Different ≥3 ≥6 Test isolate is unrelated to the outbreak

*Compared with the outbreak strain.

method involves cutting DNA with restriction enzymes, much easier to interpret. Although the method is limited
resolving the resulting fragments by gel electrophoresis, by the sequences that can be amplified and differenti-
and then transferring the separated fragments to a mem- ated through restriction enzyme digestion, proper gene
brane for probing with a specific probe. Gene-specific selection provides a highly reproducible and discrimi-
probes are used to identify or subtype microorganisms natory test. In one study, an 820-bp amplified fragment
such as P. aeruginosa in patients with cystic fibrosis and of the ureC gene from Helicobacter pylori digested with
nosocomial L. pneumophila infections. Sau3A and Hhal yielded 14 different Sau3A patterns and
For strain typing of M. tuberculosis by RFLP, the 15 different Hhal patterns. These patterns were informa-
probe is complementary to an insertion element, IS6110, tive as to the antibiotic sensitivity of the various types to
and will bind to restriction fragments on the membrane clarithromycin therapy.
that contains it, resulting in a series of bands that are
easily analyzed and compared. Insertion elements (inser- Arbitrarily Primed PCR
tion sequences) are segments of DNA that move inde-
Arbitrarily primed PCR, or random amplified poly-
pendently throughout the genome and insert themselves
morphic DNA (RAPD) assay, is a modified PCR using
in multiple locations. Strains can be RFLP-typed based
short (e.g., 10-base-long) oligonucleotides of random
on how many insertions are present and where they are
sequences to prime DNA amplification all over the
located. Isolates from the same strain will have the same
genome. The gel pattern of amplicons produced is char-
number and location of elements.
acteristic of a given organism. If two organisms have the
The gene targets selected for RFLP depend on the
same pattern, they are considered the same type; if the
organism under investigation and which genes will be
patterns differ, they are different types. The RAPD assay
most informative. Ribosomal RNA genes are highly
is relatively fast and inexpensive; however, producing
informative over a range of microorganisms, which is
consistent results may be technically demanding. Accu-
the basis for a modification of the RFLP procedure,
rate interpretation of RAPD raw data requires that the
which is a form of ribotyping. For this method, probes
procedure conditions be followed strictly so that pattern
target the 16S and 23S rRNA genes. RFLP and ribotyp-
differences (not necessarily patterns) are reproducible
ing have been applied in industrial as well as clinical
(Fig. 11.12).
microbiology.
RFLP can be performed more rapidly using PCR
Amplified Fragment Length Polymorphism
amplification with gene-specific primers (locus-specific
(AFLP) Assay
RFLP [PCR-RFLP]). This method requires the ampli-
fication of specific regions by PCR. The amplicons are AFLP is a name, rather than an acronym, chosen by the
then cut with restriction enzymes, yielding bands of in- inventors of this assay due to its resemblance to RFLP.
formative size. An advantage of this procedure, in addi- The AFLP assay is based on the amplification of DNA
tion to its speed, is the simple banding pattern, which is fragments generated by cutting the test genome with
 Chapter 11 • Detection and Identification of Microorganisms 333

M C

A
M C

FIGURE 11.12 RAPD gel results. An unacceptable

gel pattern is represented in panel A. The bands are
smeared, and variable producing patterns are too
complex for positive identification of unrelated
strains. The gel pattern represented in panel B is
acceptable. Strain differences can be clearly identi-
fied by variations from the known strain (C). Molec-
ular-weight markers are shown in lane M. B

restriction enzymes. DNA isolated from the test strain transposable elements (stretches of DNA that move from
is digested with HindIII or other restriction enzyme one location to another in a non-Mendelian fashion; also
(Fig. 11.13). Short DNA fragments or adaptors are called jumping genes). The genomic locations of these
ligated to the ends of the cut DNA. The adaptor-ligated structures are related to species type and can be used to
fragments are then amplified in two steps with primers distinguish between bacterial isolates.
complementary to the adaptor sequences. Nucleotides Enterobacterial repetitive intergenic consensus
located at the 3′ end of the primers select for specific (ERIC) sequences are 126-bp-long genomic sequences
sequences in the restriction fragments. The amplicons found in some bacterial species that are highly con-
are then resolved by gel or capillary electrophoresis (flu- served, even though they are not in coding regions.
orescently labeled primers are used for capillary electro- These sequences are located between genes in operons
phoresis). The pattern will be characteristic of the strain or upstream or downstream of single open reading
or type of organism. This assay can be performed with frames. ERIC sequences are flanked by inverted repeats
one or two enzymes (e.g., EcoR1/MseI or BamH1/PstI). that could form stem-loop or cruciform structures in
AFLP may detect more polymorphisms than RAPD DNA and are found only in gram-negative organisms,
analysis and is faster than PFGE. The procedure is more such as Bartonella, Shigella, Pseudomonas, Salmonella,
technically demanding, however, than a similar method, Enterobacter, and others.
REP-PCR (discussed in the next section). Gel patterns A related type of repetitive element, the repetitive
may also be complex (Fig. 11.14). Both high and low extragenic palindromic (REP) sequence, is similar to the
reproducibility for the method have been reported. ERIC sequence in that it occurs in noncoding regions
and contains an inverted repeat. REP sequences differ
from ERIC sequences in size (REP sequences are only
Interspersed Repetitive Elements
38 bp long), in being more numerous in the genome, and
Copies of conserved sequences are found through- in being present in multiple copies at a single location
out the genomes of most organisms. These sequences (Fig. 11.15). PCR primed from these elements yields a
may have arisen from viral integration or movement of series of products that can be resolved by gel, capillary
334 Section III • Techniques in the Clinical Laboratory

Chromosomal DNA
HindIII fragment
5′ AAG CTT A 3′
3′ A TTC G A A 5′

Ligate adaptors

Adaptor Adaptor
5′ AAG CTT AAGCTT 3′
3′ TTCG A A TTC G A A 5′

Amplify with
preselective primers FIGURE 11.13 AFLP analysis begins with
restriction digestion of chromosomal DNA.
N
The resulting fragments (top) are ligated with
5′ AAG CTT AAGCTT 3′ adaptors compatible with the restriction
3′ TTCG A A TTC G A A 5′ enzyme ends and complementary to primers
N used to amplify them. The first amplification is
Amplify with performed with preselective primers that end
selective primers
in a 3′ base (N) selected by the user. Selective
NNN primers with three added 3′ bases are used for
5′ AAG CTT AAGCTT 3′ a second round of PCR. This selection results
3′ TTCG A A TTC G A A 5′ in a characteristic pattern; only a fraction of
the original fragments will be represented in
NNN
the gel pattern.

electrophoresis, or microfluidics into characteristic pat- is located between the 5.8S and the 28S rRNA genes.
terns (Fig. 11.16). These elements have been used for Two additional elements, intergenic spacer (IGS)
typing of clinically important organisms, such as Clos- regions, IGSI and IGSII, are located between the
tridium difficile and fungal pathogens. rDNA repeat units (Fig. 11.17). Used for the identifi-
Another repetitive element, BOX, was discovered in cation and typing of yeast and molds, ITS sequences
S. pneumoniae. BOX elements consist of different com- are conserved within species but polymorphic between
binations of subunits, boxA, boxB, and boxC, which species. The ITS sequences are amplified using primers
are 59, 45, and 50 bp long, respectively. Although these directed to the unique 17S and 26S gene sequences. The
elements are not related to ERIC and REP sequences, resulting amplicons are analyzed by sequencing, sin-
they do form stem-loop structures, as do ERIC and REP. gle-strand conformation polymorphism, density-gradi-
About 25 of these elements are present in the S. pneumo- ent gel electrophoresis, restriction enzyme analysis, or
niae genome, where they may be involved in regulation sequence-specific PCR.
of gene expression.
spa Typing
Internal Transcribed Spacer Elements
MRSA contains a VNTR element in the 3′ coding region
The ribosomal RNA genes comprise the most con- of the protein A gene (spa). The element consists of repeat
served region in the genome. These genes are arranged units of 21 or 24 bp in length. Repeat units also vary by
as an operon, including a small subunit, 18S rRNA, sequence of at least one position. The spa element can
5.8S rRNA, and a large subunit, 28S rRNA. The ITS 1 have 2 to 16 repeat units. Analysis of these elements by
and 2 elements (ITS1 and ITS2) are found in regions PFGE or sequencing and comparison to known isolates
separating the 18S and the 28S rRNA genes. ITS1 is (spa typing) is used to identify MRSA. Almost 400
located between the 18S and the 5.8S gene, and ITS2 repeat profiles or spa types have been defined.
 Chapter 11 • Detection and Identification of Microorganisms 335

A similar sequence structure in the coagulase this method is increased by analysis of other repeated
gene (coa) has also been used for S. aureus typing sequences elsewhere in the MRSA genome. The com-
(coa typing). The coa VNTR units are 81 bp in length. bination of spa and VNTR typing has a discrimina-
PCR amplification and sequence analysis are used tory power equal to that of PFGE, with a more rapid
to analyze coa types. The discriminatory power of turnaround time.

1 1 2 2 3 3 Isolate A

Isolate B

M A B M A B U

FIGURE 11.16 Species identification by REP or ERIC

primed amplification. REP and ERIC sequences are present in
different chromosomal locations in bacterial subtypes A and B.
PCR, which extends outward-oriented primers hybridized to
the repeated sequences, generates amplicons of different sizes
based on the placement of the element, as in the gel depicted
in the bottom left panel where only one amplicon is shown.
FIGURE 11.14 Banding patterns generated by fluorescent Multiple REPs and ERICs throughout the bacterial chromo-
AFLP analysis. Note that duplicate specimens (1, 2, and 3) do some generate multiple amplicons with characteristic gel pat-
not produce the exact pattern because of band shifts and differ- terns (bottom right). An unknown isolate (U) is identified as
ent band intensities. the same strain as isolate B. M, molecular-weight markers.

ERIC sequence

…GTGAATCCCCAGGAGCTTACATAAGTAAGTGACTGGGGTGAGCG…

REP sequence

…GCC G/T GATGNCG G/A CG C/T NNNNN G/A CG C/T CTTATC C/A GGCCTAC…

FIGURE 11.15 The central inverted repeat of a 126-bp ERIC sequence (top) and a consensus REP sequence (bottom). ERIC
sequences contain multiple inverted repeats (arrows) that can generate a secondary structure consisting of several stems and loops.
REP sequences contain a conserved inverted repeat that forms a stem with a loop that includes 5 bp of variable sequence (N).
336 Section III • Techniques in the Clinical Laboratory

One rRNA repeat unit

rRNA
SSU (18S) ITS1 5.8S ITS2 LSU (28S) IGS1 5S IGS2 SSU (18S) ITS1 5.8S ITS2 LSU (28S) IGS1 5S

FIGURE 11.17 Ribosomal RNA genes are arranged in multiple tandem units that include the major rRNA transcript (18S to 28S)
and the 5S gene. ITSs are located within the major transcript template area and IGSs in the region between the repeat units sur-
rounding the 5S rRNA gene.

MALDI-TOF peak patterns or peptide mass profiles.68

TABLE 11.13 Example of Housekeeping Genes The profiles are analyzed by searching for matching
Sequenced in an MLST Test profiles in extensive open-source and intralaboratory
databases. MALDI methods are faster than some genetic
Number methods and can successfully detect organisms from
Gene Gene Product of Alleles
mixtures. Use of MALDI results for stain comparisons
ArcC Carbamate kinase 52 is limited, however. Peptide profiles are influenced by
growth conditions which affect microbial physiol-
AroE Shikimate dehydrogenase 88 ogy and protein expression. Although identification by
GlpF Glycerol kinase 55 mass spectrometry requires culture of microorganisms,
culture conditions and time of culture do not seem to
Gmk Guanylate kinase 51 alter profiles.69,70
Pta Phosphate acetyltransferase 57 Speciation of organisms that cause environmen-
tal hazards, such as food- and water-borne diseases, is
Tpi Triosephosphate isomerase 74 important not only for investigating but also for prevent-
YqiL Acetyl coenzyme A acetyltransferase 66 ing outbreaks. Only some strains of organisms are impli-
cated in outbreaks. Peptide maps of organisms, such as
Aeromonas, from which 7 of 17 species are related to
Multilocus Sequence Typing water outbreaks, allow accurate classification of species
for environmental monitoring.71 MALDI has also been
Multilocus sequence typing (MLST) characterizes bac- applied to the identification of food pathogens such as
terial isolates by using sequences of internal fragments lactic acid bacteria from spoiled food, toxin-producing
of housekeeping genes. Six or seven genes are sequenced bacteria responsible for food poisoning, and beneficial
over 450 to 500 bases, and the sequences are assigned bacteria in probiotics and yogurt.
as alleles. Examples of housekeeping genes used in Whole-cell MALDI-TOF mass spectrometry can be
S. aureus MLST are shown in Table 11.13. Distinct used to identify and compare organisms from highly
alleles are defined as single-base-pair differences or contaminated ecosystems, such as soil and sewage
multiple changes resulting from recombination or other sludge.72 In this application, intact cells are extracted
genetic events. An isolate type or the allelic profile is the with trifluoroacetic acid and acetonitrile and permeabi-
collection of all seven alleles, also called the sequence lized with glass beads before ionization. This method is
type. Bacteria have enough variation so that there are also applied to intact conidia and spores for identifica-
multiple alleles of each housekeeping gene, making up tion of fungal organisms.
billions of allelic profiles. Because the data generated by
MLST is text sequence, the results can be compared with
those in large databases, for example, http://pubmlst.org. Comparison of Typing Methods
Genotypic methods used for strain typing are evaluated
Mass Spectrometry
and compared based on five criteria. First, the target
Peptides (2 to 20 kd) of the highly abundant ribo- organism must be typable by the method used to detect
somal proteins are assessed to identify microbes by it (typing capacity). A test that detects a genotypic or
 Chapter 11 • Detection and Identification of Microorganisms 337

TABLE 11.14 Performance Comparison of Representative Molecular Epidemiology Methods*

Typing Discriminatory Ease of

Method Capacity Power Reproducibility† Ease of Use Interpretation‡

Plasmid analysis Good Good Good High Good

PFGE High High High Moderate Good–Moderate

Chromosomal RFLP High Good Good High Moderate–Poor

Ribotyping High High High Good High

PCR-RFLP Good Moderate Good High High

RAPD High High Poor High Good–High

AFLP High High Good Moderate High

Repetitive elements Good Good High High High

Sequencing High High High Moderate Good–High

Mass spectrometry High Good-High Good Good High

*From Olive and Bean.76

†
Intralaboratory
‡
Interpretation is influenced by the quality of the data.

phenotypic characteristic that is not expressed in all interpretation. Unfortunately, no such method fits this
members of a species will not accurately detect the profile, so the laboratory professionals performing these
target organisms at all times. A molecular assay must types of analyses may have to sacrifice ease of perfor-
target a reasonably polymorphic DNA sequence, alleles mance, for example, in order to get excellent discrimi-
of which are unambiguously associated with a given natory power when they are choosing which molecular
strain. Second, the method must be reproducible. A typing method to use.
reproducible method yields the same result on repeated In conclusion, molecular-based methods are available
testing of the same organism or strain. Variations in cell for the detection, identification, and characterization of
characteristics, such as antigens or receptor expression, a number of microorganisms. For some, assays are used
decrease reproducibility (precision). Third, the method almost exclusively, such as for N. gonorrhoeae, C. tra-
must clearly distinguish between unrelated strains (dis- chomatis, and B. pertussis. For others, molecular-based
criminatory power). Reproducibility and discriminatory tests are used to provide rapid results and supplement
power are important for the establishment of databases traditional testing, such as for M. tuberculosis. Finally,
that can be used by independent laboratories. Fourth, molecular-based methods are undergoing continuous
ease of interpretation of results is important. Unclear development and experiencing increasing use compared
or complex results will lower reproducibility and dis- to traditional culture and phenotypic methods.
criminatory power. Finally, ease of test performance is
important in order to minimize the chance of error or
ambiguous results. A rating of representative methods is Case Study 11.1
shown in Table 11.14.
The most desirable typing method is the one that During a holiday weekend at a luxury hotel, guests
will type all strains and have excellent reproducibil- began to complain of stomach flu with nausea and
ity, discriminatory power, and ease of performance and
338 Section III • Techniques in the Clinical Laboratory

vomiting. In all, more than 100 of the 200 guests Case Study 11.2
who had dined at the hotel the previous evening
described the same symptoms. Eight people had Five students from different local community
symptoms severe enough to warrant hospital- high schools suffered recurrent skin infections
ization. Most, however, recovered within 24 to with chronic wounds. Nasal swabs and skin
48 hours of the onset of symptoms. Health offi- specimens from the students were screened for
cials were notified. Interviews and epidemiological MRSA by inoculation onto Mueller–Hinton agar
analyses pointed to a Norwalk-like virus infection, supplemented with NaCl (0.68 mol/L) containing
or norovirus, probably foodborne. Stool specimens 6 μg/mL of oxacillin. The cultured organisms
(1 to 5 mL) from hospitalized patients and samples exhibited an MIC of more than 32 μg/mL vanco-
of the suspected food sources (500 mg) were sent mycin and a zone of inhibition of less than 14 mm
for laboratory analysis by RT-PCR. RNA extracted in diameter. Isolates were sent to the CDC and
with 1,1,2 trichloro-1,2,2, trifluoroethane was referred for molecular testing. DNA isolated from
mixed with a guanidium thiocyanate buffer and iso- the five cases and a control strain were embedded
lated by organic (phenol-chloroform) procedures. in agarose plugs and digested with SmaI for PFGE
cDNA was synthesized using primers specific to analysis. A depiction of the gel pattern is shown in
the viral RNA polymerase gene, and strain-specific the accompanying figure.
PCR primers were used to amplify the viral gene. M 1 2 3 4 5 6 7 M
The amplicons, resolved by agarose gel electro-
phoresis, are shown in the accompanying figure.

PFGE patterns of isolated strains. M, molecular-weight

markers. Lanes 1 to 5, isolates from the community infections;
lanes 6 and 7, unrelated hospital isolates.

RT-PCR products were resolved on separate gels. Amplicons Further PCR testing was performed on the iso-
from four affected individuals (lanes 1 to 4, left). Lane 5, posi- lates for virulence factors, particularly for mecA
tive control; lane 6, sensitivity control; lane 7, negative control;
lane 8, reagent blank. Specimens from suspected food sources
gene sequencing and detection of the Panton–
(lanes 1 to 4, right). Lane 5, salad lettuce from the distributor; Valentine leukocidin (PVL) genes, lukS-PV and
lanes 6 to 8, specimens from three hotel employees working lukF-PV. The results from these tests revealed that
the day of the outbreak; lane 9, molecular-weight marker. all five isolates contained the PVL genes and the
type IV mecA element.
QUESTIONS:
QUESTIONS:
1. Do the four patients have norovirus?
1. Are all or some of the five isolates the same
2. What is the source of the organism?
or different? Which isolates are the same, and
3. When did the source get contaminated? Did the
which are different? What is the evidence to
source come into the hotel infected, or was it
support your answer?
infected inside the hotel?
2. Was there a single source for these organisms
or multiple sources?
 Chapter 11 • Detection and Identification of Microorganisms 339

c. Ribotyping
Case Study 11.3 d. Bacteriophage typing

A 39-year-old HIV-positive male has been moni- 3. For which of the following organisms must caution
tored closely since his diagnosis as HIV-positive be exercised when evaluating positive PCR results
5 years ago. The man was on HAART and com- because the organism can be found as normal flora
pliant. He was relatively healthy and had not even in some patient populations?
had a cold in the last 4 years. The man had HIV
viral loads as determined by Amplicor RT-PCR a. Neisseria gonorrhoeae
that were consistently close to 10,000 copies/mL, b. HIV
never varying more than 0.2 log10 unit, until the c. Chlamydophila pneumoniae
last 6 months, when his viral loads were trending d. Streptococcus pneumoniae
up to 25,500, then 48,900, with his most recent
result being 55,000 copies/mL. Genotyping per- 4. Which of the following controls are critical for
formed on virus isolated from the patient revealed ensuring that amplification is occurring in a patient
a mutation in the reverse transcriptase gene of sample and that the lack of PCR product is not due
M41L that is associated with resistance to zidovu- to the presence of polymerase inhibitors?
dine (AZT). a. Reagent blank
QUESTIONS: b. Sensitivity control
1. What is the significance of the viral load results c. Negative control
over the last 6 months? d. Amplification control
2. What is the implication of the genotyping result
for the patient's therapy? 5. A PCR assay was performed to detect Bordetella
3. How should this patient be monitored in the pertussis on sputum obtained from a 14-year-old
future? girl who has had a chronic cough. The results
revealed two bands, one consistent with the
internal control and the other consistent with
the size expected for amplification of the
B. pertussis target. How should these results
be interpreted?
STUDY QUESTIONS a. These are false-positive results for B. pertussis.
b. The girl has clinically significant B. pertussis
infection.
1. Which of the following genes would be analyzed
c. B. pertussis detection is more likely due to
to determine whether an isolate of Staphylococcus
colonization.
aureus is resistant to oxacillin?
d. The results are invalid because two bands were
a. mecA present.
b. gyrA
c. inhA 6. Which of the following is an advantage of
d. vanA molecular-based testing?
a. Results stay positive long after successful
2. Which of the following is a genotypic method
treatment.
used to compare two isolates in an epidemiological
b. Results are available within hours.
investigation?
c. Only viable cells yield positive results.
a. Biotyping d. Several milliliters of specimen must be
b. Serotyping submitted for analysis.
340 Section III • Techniques in the Clinical Laboratory

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