Nucleic Acid Amplification Tests for the Diagnosis of Pneumonia
CID
Nucleic Acid Amplification Tests for the Diagnosis of Pneumonia
L. Barth Reller 0
Melvin P. Weinstein 0
Section Editors 0
0 David R. Murdoch Microbiology Unit , Canterbury Health Laboratories , and Department of Pathology, Christchurch School of Medicine and Health Sciences , Christchurch , New Zealand
Molecular diagnostic techniques, such as polymerase chain reaction (PCR), are promising tools for the rapid etiological diagnosis of pneumonia. PCR offers potential advantages over conventional tests for the detection of Mycoplasma pneumoniae, Legionella species, and Chlamydia pneumoniae. For pneumococcal pneumonia in adults, PCR adds little to existing diagnostic tests and is unable to distinguish pneumococcal colonization from infection when testing respiratory samples. Although PCR is probably more sensitive than are conventional microscopy-based methods for diagnosing Pneumocystis carinii pneumonia, the specificity is uncertain, because P. carinii can occasionally be detected in the absence of clinical symptoms. PCR is useful for the diagnosis of viral pneumonia in immunocompromised patients. Further work is required to better characterize the role of PCR versus the role of other tests for diagnosing pneumonia and to develop standard PCR assays that can be readily adopted by routine diagnostic laboratories.
-
Pneumonia is one of the most common infectious diseases
among adults and children. Despite recent advances in
diagnostic testing, the etiological diagnosis of pneumonia is
infrequently achieved with confidence. Even in the most rigorous
studies, it is difficult to establish an etiological diagnosis in 50%
of cases of community-acquired pneumonia [
1
]. In the real
world, the diagnostic rate is much lower. This has led some
investigators to question the usefulness of routinely performing
microbiologic tests for patients with pneumonia [
2–4
].
Although there may be some justification for not routinely
ordering microbiologic tests for every patient with pneumonia,
it is important to recognize recent advances in diagnostic
testing. The most promising advances have been with antigen and
nucleic acid detection assays. New immunochromatographic
urinary antigen detection assays for Legionella pneumophila and
Streptococcus pneumoniae are easy and quick to perform and
have relatively high sensitivities and specificities [
5, 6
].
Moreover, with further refinement and development, it is expected
that many other antigen detection tests for pneumonia
path
MOLECULAR DIAGNOSTIC TECHNIQUES
FOR PNEUMONIA
PCR is an attractive tool for diagnosing the cause of pneumonia,
because it can detect minute amounts of nucleic acid from
potentially all pneumonia pathogens, does not depend on the
viability of the target microbe, is probably less affected by
previous antimicrobial therapy than are culture-based methods,
and provides results quickly. Moreover, increased
mechanization means that PCR is becoming increasingly available to
laboratories outside of specialist tertiary referral centers. The
development of real-time PCR technology enables testing to be
performed in !1 h in a single reaction vessel, thereby reducing
the chance of contamination. These advantages may make PCR
the front-runner for the ideal diagnostic test for pneumonia
[
9
], although there are some disadvantages of relying on
molecular approaches rather than culture. Such disadvantages
include the limited ability to perform antimicrobial susceptibility
testing and the lack of an isolate archive if future testing is
required.
As yet, no PCR assay has been approved by the US Food
and Drug Administration for diagnosis of pneumonia, although
commercial assays are becoming available. For this to happen,
optimal protocols need to be established. Several key
parameters need to be determined before PCR becomes part of the
routine diagnostic workup for a particular pneumonia
pathogen, notably sensitivity and specificity, reproducibility, and
optimal sample types.
SENSITIVITY AND SPECIFICITY
The accurate calculation of clinical sensitivity for a PCR assay
is hindered by the lack of a suitable diagnostic “gold standard”
for most infections. This is an inherent problem with PCR,
which is likely to be a more sensitive technique than are most
culture-based diagnostic tests. Sensitivity may be affected by
the presence in samples of PCR inhibitors, usually those of an
unknown nature, which cause false-negative results [
10
]. These
inhibitors can be detected by the inclusion of specific positive
internal controls, which should be part of every PCR assay.
Given the small volumes of PCR reaction mixtures, sampling
errors may also reduce sensitivity, which may be improved by
increasing the sample volume in the reaction mixture or
through concentration of the sample.
The risk of false-positive results is a major problem facing
all PCR assays. This is largely a consequence of the extreme
sensitivity of PCR and may result from contamination by
exogenous (...truncated)