Infectous Disease Clinics of N. America [Journal, Vol 20] (Elsevier, 2006) WW.pdf

Infect Dis Clin N Am

20 (2006) 735–758

Clinical Applications of the Polymerase

Chain Reaction: An Update

Raquel Cuchacovich, MD a , b

a Section of Rheumatology, Department of Medicine, Louisiana State University Health

Sciences Center, 1542 Tulane Avenue, New Orleans, LA 70112, USA

b Ochsner Clinic Foundation, 1514 Jeﬀerson Highway, New Orleans, LA 70121, USA

The development, in the past decade, of nucleic acid ampliﬁcation (NAA)

and detection methods has been found to be useful in the study of the etio-

pathogenesis, diagnosis, and management of a variety of clinical (including

rheumatologic) disorders. A variety of molecular techniques have been de-

veloped that allow the ampliﬁcation and detection of minute amounts of nu-

cleic acid sequences from tissues or body ﬂuids. These NAA methods can

create millions of identical copies of DNA or RNA sequences present in

clinical samples. The use of molecular technology has dramatically changed

the approach to the laboratory diagnosis of many diseases. For example,

these methods have been useful in the diagnosis of genetic disorders, such

as sickle cell anemia, b -thalassemia, and cystic ﬁbrosis. The recent develop-

ment of NAA technology has also had signiﬁcant impact on the diagnosis

and management of many infectious diseases.

An association between infectious agents and rheumatic disorders has

been established through such methods as polymerase chain reaction

(PCR). Some rheumatic disorders are induced by viruses, such as parvovirus

B19 (erosive rheumatoid arthritis [RA]); rubella (acute and chronic arthrop-

athies); alpha viruses (acute infectious illness with fever, rash, arthritis, my-

algia, encephalitis, epidemic polyarthritis, and chronic polyarthritis);

adenoviruses (symmetric polyarthritis or a recurrent chronic polyarthritis);

coxsackievirus (symmetric polyarthritis of small joints); hepatitis B (polyar-

teritis nodosa, cryoglobulinemia, transient arthritis); hepatitis C (type II

Portions of this article originally appeared in Cuchacovich R, Quinet S, Santos AM.

Applications of polymerase chain reaction in rheumatology. Rheumatic Disease Clinics of

North America 2003;29(1):1–20.

Section of Rheumatology, Department of Medicine, Louisiana State University Health

Sciences Center, 1542 Tulane Avenue, New Orleans, LA 70112, USA.

E-mail address: rcucha@hotmail.com

doi:10.1016/j.idc.2006.09.003

id.theclinics.com

736

CUCHACOVICH

cryoglobulinemia, erosive RA, and polymyositis dermatomyositis); human

T-cell lymphotropic virus type I–associated arthritis, polymyositis, Sjo ¨ gren’s

syndrome, and RA); HIV-1 (reactive arthritis, psoriatic arthritis, spondy-

loarthropathy, and inﬁltrative lymphocytosis syndrome, and in some

cases remission of systemic lupus erythematosus, RA, polymyositis).

Gram-positive and gram-negative bacteria, such as Streptococcus sp, Chla-

mydia trachomatis, Salmonella sp, and Shigella sp have been found as trig-

gering agents for rheumatic fever and reactive arthritis [1–13] .

Several strategies for the ampliﬁcation of nucleic acids have been de-

scribed, including ampliﬁcation of the nucleic acid target (eg, PCR, strand-

displacement ampliﬁcation, self-sustaining sequence replication); ampliﬁca-

tion of a nucleic acid probe (eg, ligase chain reaction, Q [b] replicase); and

signal ampliﬁcation (eg, branched-probe DNA assay). As these molecular

methods are further reﬁned and become more widely available in the next

few years, physicians need to understand their clinical applications and be

aware of their potential advantages, limitations, and clinical usefulness.

This article describes the principles behind PCR-based diagnosis and up-

dates its clinical applications. It is beyond the scope of this article, however,

to describe other NAA methods or to include a complete list of all PCR as-

says that have been developed; other recent reviews oﬀer additional details.

Polymerase chain reaction

Molecular technologies have increased the speed of antigen detection

methods. The most widely used of these methods is PCR, a technique that

enables the ampliﬁcation of speciﬁc sequences of nucleic acids. The tech-

nique was originally described by Saiki and coworkers and subsequently per-

fected by Mullis in 1987. PCR can amplify minute amounts of target DNA

(10 to 100 copies in clinical samples) within a few hours. In the laboratory

PCR is used for DNA sequencing, cloning, gene isolation, and analysis of

gene expression, and for sequencing of mitochondrial and genomic DNA

in the human genome organization project. PCR can be combined with

other techniques to determine whether the ampliﬁcation products contain

a mutation, such as restriction enzyme digestion, allelic speciﬁc oligonucle-

otide hybridization, and single-strand conformation polymorphism analysis.

Applications in microbiology and infectious diseases have included the

diagnosis of infection caused by slow-growing or fastidious microorganisms;

detection of infectious agents that cannot be cultured; presence of novel mi-

croorganisms (Tropheryma whippelii) [14] ; and recognition of newly emerg-

ing infectious diseases (nearly 100 kinds of organisms have been detected by

PCR). The procedure also improves the accuracy of subtyping pathogens in

epidemiologic studies, quantiﬁes the viral load [15] , and allows rapid identi-

ﬁcation of antimicrobial resistance. PCR can also be used to detect RNA

viruses [16–18] (eg, hepatitis C virus); a speciﬁc messenger RNA (mRNA)

transcribed by a microorganism; DNA virus in autoimmune conditions

UPDATE ON PCR CLINICAL APPLICATIONS

737

treated with immunosuppressive drugs [19–21] ; and to identify cases of HIV

patients with rheumatologic conditions ( Box 1 ). It is important to under-

stand how PCR-based techniques are used to detect the presence of infec-

tious agents in which there are too few organisms present for detection by

other means. This is illustrated by the use of PCR for the detection of My-

cobacterium infection, especially tuberculosis in RA patients treated with tu-

mor necrosis factor- a inhibitors. Tuberculosis among RA patients before

the use of tumor necrosis factor- a inhibitors was approximately 6 cases

Box 1. Applications of PCR

Genetics

Detection of genetic defects associated with inherited diseases

Detection of mutations associated with genetic diseases

Determination of genetic susceptibility to a disease

Determination of disease risk to offspring in families with

affected members

Detection of cancer and determination of the extent of residual

disease

Ability to detect clonality with a high sensitivity (0.001%–0.1%)

Detection of gene polymorphism

Detection of nonself cells or occult neoplastic cells in tissues

Detection of genetic markers (receptor cell rearrangements,

major histocompatibility complex system)

Forensic determination of identity

Infectious diseases

Ability to diagnose infections caused by slow-growing or

fastidious microorganisms

Detection of infectious agents that cannot be cultured or

organisms that have not yet been identiﬁed

Specie identiﬁcation in the mycobacterium

Useful for screening, diagnosis, and management of viral

infections (hepatitis viruses, human herpes virus 8, HIV)

Recognition of newly emerging infectious disease

Detection of RNA viruses (eg, hepatitis C virus) or speciﬁc mRNA

transcribed by a microorganism

Recognition of viral load to monitor therapy

Detection of infections in autoimmune conditions treated with

immunosuppressive drugs

Diagnosis of viral encephalitis

Identiﬁcation of bacterial DNA for the diagnosis of septic arthritis

and reactive arthritis

Allows rapid identiﬁcation of antimicrobial resistance

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CUCHACOVICH

per 100,000 patients; it has increased to 24 cases per 100,000 patients follow-

ing the institution of tumor necrosis factor- a inhibitors. Tuberculosis has

been reported with the use of all tumor necrosis factor- a inhibitors including

etanercept, inﬂiximab, and adalimumab. It is frequently caused by reactiva-

tion of latent tuberculosis, or new infection with Mycobacterium avium and

M avium-intracellulare. These nontuberculous mycobacteria have overlap-

ping phenotypic properties that make their speciation dicult to determine

by conventional methods; also, their clinical picture can be atypical with iso-

lated fever, and these presentations may lead to delays in the diagnosis with

subsequent dissemination. Smear can be negative 50% of the time, particu-

larly in immunosuppressed individuals; cultures can take a long time, which

makes the diagnostic approach problematic. PCR can provide both rapid

results and an improved diagnostic accuracy of the involved mycobacteria

(Mycobacterium tuberculosis from nontuberculous mycobacteria) in the dis-

ease process, and lead to the right therapeutic approach in a short period of

time [8,11,22] .

PCR is also used to detect mutations associated with genetic diseases, for

tissue typing, and to diagnose neoplastic diseases. It is also a valuable tool

for ampliﬁcation of T-cell receptor genes to distinguish neoplastic prolifer-

ation from reactive T cells. Moreover, many diseases not traditionally

thought to be genetic are now being evaluated in terms of inherited suscep-

tibilities, or as genetically mediated maladaptive responses to external stim-

uli, such as autoimmune diseases.

PCR also facilitates antibody engineering, such as monoclonal drugs an-

tibodies. The isolation of an individual antigen-binding B cell is sucient to

isolate the relevant antibody-binding variable (V) region, by using oligonu-

cleotide primers to amplify antibody V regions. In addition, the V region of

the monoclonal antibody can be fused to the constant region to produce the

desired isotype and subclass; by this method the entire antibody repertoire

in the form of recombinant antibody libraries can be obtained (see Box 1 ).

In the past few years a variant of this technique, called broad-range PCR,

has enabled researchers to identify a number of uncultivable microbial path-

ogens. This approach, which uses ribosomal RNA (rRNA) gene sequences

(which are found in all prokaryotes and eukaryotes and contain highly con-

served regions interspersed with species-speciﬁc ones), makes it possible to

perform PCR on infected tissue containing extremely small numbers of

bacteria.

First, the highly conserved regions of the bacterial rRNA gene are used to

‘‘prime’’ the synthesis of the remainder of the gene. The resulting PCR am-

pliﬁed product is then sequenced to identify bacteria-speciﬁc variable re-

gions of the gene. Such an approach has enabled researchers to identify

a variety of microbes, including the etiologic agents of bacillary angiomato-

sis and Whipple’s disease.

An enzymatic in vitro DNA method PCR can be coupled with a number

of other detection methods to identify any gene (by DNA) or its expression

UPDATE ON PCR CLINICAL APPLICATIONS

739

(by mRNA) directly from clinical samples, regardless of the amount of tar-

get molecules present [23] . This application requires reverse transcription

(RT) of the RNA isolated from the sample into DNA; the product of this

reaction, termed ‘‘complementary DNA’’ (cDNA), then serves as the tem-

plate for ampliﬁcation by PCR. Caution must be exercised in these cases

because RNA is a labile molecule and its degradation could lead to false-

negative results. This process depends on a uniquely synthesized pair of

oligonucleotide primers that ﬂank and deﬁne the DNA segment of interest.

In the initial step of the procedure, nucleic acid (eg, DNA) is extracted from

the microorganism or clinical specimen of interest. A thermally stable DNA

polymerase uses the target DNA strand to which the primer has bound as

a template to synthesize a complementary strand of DNA in an automated

instrument known as a ‘‘thermocycler.’’ Heat (90 C–95 C) is used to sepa-

rate the extracted double-stranded DNA into single strands (denaturation).

Cooling to 55 C then allows primers speciﬁcally designed to ﬂank the target

nucleic acid sequence to adhere to the target DNA (annealing). The enzyme

Taq polymerase and nucleotides are then added to create new DNA frag-

ments complementary to the target DNA (extension). This completes one

cycle of PCR. This process of denaturation, annealing, and extension is

repeated numerous times in the thermocycler. At the end of a cycle, each

newly synthesized DNA sequence acts as a new target for the next cycle,

so that after 30 or more cycles, millions of copies of the original target

DNA are created. The result is the accumulation of a speciﬁc PCR product

with sequences located between the two ﬂanking primers. Repeated cycles

result in a 2n exponential increase of the template DNA, where n is the

number of cycles. Detection of the ampliﬁed products can be done by visu-

alization with agarose gel electrophoresis, an enzyme immunoassay format

using probe-based calorimetric detection, or by ﬂuorescence emission

technology.

A major strength of PCR is that the DNA segment of interest does not

need to be puriﬁed from the background DNA. Furthermore, PCR can

be used to diagnose disease using almost any tissue or body ﬂuid, including

fresh and archival specimens. The exponential ampliﬁcation of PCR pro-

vides for great sensitivity and the use of unique primers provides for the

speciﬁcity. Unlike other assay systems in which sensitivity is often compro-

mised for the sake of speciﬁcity, an increase in one of these parameters leads

to increases in the other [24,25] .

Variations on the standard polymerase chain reaction

Multiplex polymerase chain reaction

In multiplex PCR the assay is modiﬁed to include several primer pairs

speciﬁc to diﬀerent DNA targets to allow ampliﬁcation and detection of

a number of diﬀerent sequences at the same time (eg, two pathogenic viruses

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