PERSPE C T I V E
FOCUS ON RESE ARCH
The New Age of Molecular Diagnostics for Microbial Agents
Richard Whitley, M.D.
H ow, in this era of molecular
diagnostic tests, can we best
determine whether there is a
causal relationship between the
presence of a genetic signature
of an infectious agent and dis-
ease? In recent years, molecular
techniques have been applied suc-
cessfully in the identification of
infectious agents such as Borna
virus, Kaposi’s sarcoma–associ-
ated herpesvirus (human herpes­
virus 8), West Nile virus, and the
severe acute respiratory syndrome
(SARS) coronavirus.1 Currently, the
majority of surveillance and dis-
covery efforts use methods based
on sequences of known agents —
namely, competitive polymerase
chain reaction (PCR) and micro-
arrays. Such efforts fail, however,
when the agents in question are
truly novel or sufficiently distant in
sequence from related agents that
they confound hybridization.
A recent case in point was
that of a febrile illness that, after
similar clinical courses, led to the
deaths of three patients who had
recently received visceral-organ
transplants from a single donor.
All three patients had identical
viral sequences, and in one of
them, seroconversion to this
agent occurred, indicating recent
exposure. As Palacios and col-
leagues report in this issue of
the Journal (pages 991–998), new
diagnostic techniques were re-
quired in order to identify what
turned out to be a new arenavi-
rus that had been transmitted
through organ transplantation.
In brief, RNA from two recipi-
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The New Age of Molecular Diagnostics for Microbial Agents
ents’ transplanted organs — a
liver and a kidney — was used as
starting material to create a li-
brary of more than 100,000 se-
quences. Bioinformatics tools
were then used to subtract hu-
man sequences and identify the
residual sequences that might
represent microbial pathogens. In
the short term, the findings of
Palacios et al. clarify some aspects
of the natural history of arena­
virus infection in immunocom-
promised patients at high risk
for infection. More significantly,
this application of new diagnostic
techniques may ultimately trans-
form microbiologic testing.
The investigators describe a
method of adapting high-through-
put DNA pyrosequencing to patho-
gen discovery and discuss the
innovations in specimen prepara-
tion and bioinformatics that per-
mitted them to identify this new
arenavirus in a cluster of cases
of fatal encephalitis. Pyro­
sequencing entails the enzymatic
incorporation of complementary
nucleotides into an elongating
DNA chain; this process results
in pyrophosphate release. A cam-
era registers this emission as
light. This method has been
adapted to a slide format, where-
in hundreds of thousands of wells
containing individual microbeads
coated with DNA are sequenced
in parallel (see diagram).
High-throughput pyrosequenc-
ing has been used primarily to
sequence large genomes; to my
knowledge, it has not previously
been applied to pathogen discov-
Related article, p. 991
ery. However, when Palacios et al.
found that the classic application
of typical diagnostic assays, in-
cluding viral culture, electron mi-
croscopy, PCR, and panmicrobial
microarrays, failed to identify an
infectious agent, they turned to
pyrosequencing. Although derived
sequences at the nucleic acid level
were not informative, bioinformat-
ic analyses at the protein level re-
vealed footprints of a potential
causative agent. Using these se-
quences as a foundation for de-
veloping specific molecular re-
agents, the investigators cloned
the remainder of the viral genome
by means of PCR, propagated it
in cell culture, defined its mor-
phologic characteristics by means
of electron microscopy, and cre-
ated molecular and serologic as-
says for detection of infection.
Such metagenomic pyro­
sequencing permits the rapid
identification of all nucleic acid
sequences in a sample. It has been
the primary method used for all
sequencing of both human and
bacterial genomes. Other appli-
cations have included studies of
microbial diversity — for exam-
ple, the sequencing of HIV and of
bacterial microflora detected in
either healthy people or those with
a given disease.2,3 Unlike compet-
itive PCR or array methods, in
which investigators are limited by
sequence information for known
organisms and must make choic-
es regarding the range of patho-
gens to be considered, meta­
genomic sequencing is unbiased
and provides the opportunity to
PERSPECTIVE
DNA-covered
microbeads
Clinical specimen sequenced
by chemiluminescence
Nucleic acid
sequences obtained
Viral genome cloned
Virus propagated in tissue culture
Infection confirmed through serologic
and molecular assays
Use of High-Throughput DNA Pyrosequencing
for Pathogen Discovery.
consider a broader spectrum of
organisms. Thus, it is ideal for
applications in which unknown
bacteria, fungi, parasites, or vi-
ruses may be involved.
n engl j med 358;10  www.nejm.org  march 6, 2008
Downloaded from www.nejm.org on March 5, 2008 . Copyright © 2008 Massachusetts Medical Society. All rights reserved.
The New Age of Molecular Diagnostics for Microbial Agents
The potential for the applica-
tion of this technique is signifi-
cant. Infections of the central
nervous system often pose diffi-
cult diagnostic dilemmas. In spite
of the Unexplained Encephalitis
Project sponsored by the Centers
for Disease Control and Preven-
tion, as many as 40% of patients
with altered mentation, fever, and
inflammation of the central ner-
vous system never receive a diag-
nosis that establishes the cause of
their illness. Acute respiratory tract
infections are a leading cause of
childhood illness and death, ac-
counting for nearly 2 million
deaths annually. Even in state-of-
the-art laboratories, an agent is
identified in only 30 to 60% of
cases. Similarly, in the United
States alone, enteric infection
COLOR FIGURE
results
Draft 2 in the 2/01/08 hospitalization of
Author Whitley
nearly
Fig #
500,000
1 adults
children
Title annually. Although the
vast
ME
majority
Hogan
of these cases re-
solve
DE without
Malina permanent sequel-
Artist KMK/SW
ae, as many
AUTHOR PLEASE asNOTE: 5000 deaths each
year are Pleaseattributed
Figure has been redrawn and type has been reset
check carefully
infections
Issue date 3/06/08 of unknown cause.4
This new application of meta­
genomic pyrosequencing may well
aid in the identification of un-
known microbial agents that cause
human disease.
Palacios et al. also expand our
knowledge of arenavirus infection
that occurs after organ transplan-
tation. Previously, two clusters of
arenavirus transmission through
solid-organ transplantation had
been reported. The prototypic
arenavirus is lymphocytic chorio-
meningitis virus (LCMV). Typi-
cally, disease attributed to LCMV
in an otherwise healthy host is
asymptomatic or associated with
a mild febrile illness; however, it
has been implicated in aseptic
meningitis in rare instances. Ac-
cording to the current report, all
three patients died 4 to 6 weeks
after transplantation following
an encephalopathic illness; this
indicates that this arenavirus may
be more virulent than others. Be-
cause of the protracted incuba-
tion period, health care providers
might easily have sought the
cause of the illness somewhere
other than the transplanted or-
gans and might have failed to
pursue and identify the new in-
fectious agent.
As contemporary molecular di-
agnostic approaches are applied to
medical conditions with poten-
tially infectious causes, meta­
genomic pyrosequencing should
permit us to expand our list of
offending pathogens. The iden-
tification of new agents, in turn,
and 160,000 may well lead to the development
of new prophylactic and therapeu-
tic interventions.
No potential conflict of interest relevant
to this article was reported.
to foodborne
This article (10.1056/NEJMp0708085) was
published at www.nejm.org on February 6,
2008.
Dr. Whitley is a professor of pediatrics, mi-
crobiology, medicine, and neurosurgery at
the University of Alabama at Birmingham,
Birmingham.
1. Lipkin WI, Briese T. Emerging tools for
microbial diagnosis, surveillance and discov-
ery. In: Lemon SM, Hamburg MA, Sparling
PF, Choffnes ER, Mack A, eds. Global infec-
tious disease surveillance and detection: as-
sessing the challenges — finding solutions:
workshop summary. Washington, DC: Insti-
tute of Medicine, 2007:177.
2. Ley RE, Knight R, Gordon JL. The human
microbiome: eliminating the biomedical/
environmental dichotomy in microbial ecol-
ogy. Environ Microbiol 2007;9:3-4.
3. Turnbaugh PJ, Ley RE, Hamady M, Fraser-
Liggett CM, Knight R, Gordon JL. The human
microbiome project. Nature 2007;449:804-10.
4. Frenzen PD. Deaths due to unknown
foodborne agents. Emerg Infect Dis 2004;
10:1536-43.
Copyright © 2008 Massachusetts Medical Society.
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