Comparison, optimization en evaluation of new and fast molecular diagnostic techniques for detection of atypical bacterial respiratory tract pathogens in clinical samples from patients with CAP.

Bacterial community-acquired pneumonia (CAP) is one of the most common
infectious diseases. CAP can be distinguished in 2 types: typical pneumonia,
caused by e.g. Streptococcus pneumoniae or Haemophilus influenzae and atypical
pneumonia caused by Mycoplasma pneumoniae, Legionella pneumophila or
Chlamydophila pneumoniae. Currently, therapeutic protocols include treatment
for atypical pneumonia only when there is a high clinical suspicion or after
exacerbation of the infection. Clinically, it is not always possible to
distinguish atypical pneumonia from atypical pneumonia. Thus, it can be that
patients receive suboptimal therapy.
Isolation by culture of atypical pathogens is laborious or impossible, e.g.
caused by transport problems and rapidly decreasing viability. For most
diagnostic laboratories it is a major challenge to diagnose these bacterial
pathogens. Current infection with M. pneumoniae, C. pneumoniae or L.
pneumophila is based on a four-fold or greater rise in antibody titer between
paired acute and convalescent-phase sera within 2 weeks.
However, a second serum specimen is necessary for reason of determining
infection status, and will delay the diagnosis with two extra weeks. Molecular
techniques may be helpful in rapid and robust diagnosis of atypical pneumonia.
Especially since real time PCR assays and (semi) automated DNA isolation from
clinical specimens has become available.

The objective of this study is to compare, evaluate and validate some
commercial molecular diagnostic techniques for atypical respiratory tract
infections.
Simultaneously, we intend to define the most relevant clinical material per
etiological agens. For this purpose, next to the standard diagnostic procedure,
an extra throatswab, a nasopharyngeal aspirate and a urine sample will be
collected. The results of the conventional diagnostics (serology) and an
in-house molecular-based target amplification technique will be compared with
the results of the new diagnostic tests, obtained from different clinical
samples.

This study follows a prospective design. Routine diagnostic assays will include
assays for detection by culture, PCR or serology of infection with respiratory
viruses, bacteria causing typical pneumonia, and, when indicated, special
techniques required for specific detection of parasites, fungi or mycobacteria.
In addition to this routine standard diagnostic workup, a range of assays will
be included to detect infection with pathogens causing atypical pneumonia:
· Strand Displacement Amplification (SDA, BDProbeTec, Beckton & Dickinson)
· Multi Ligase Probe-mediated Amplification (MLPA, Pathofinder)
· Nucleic Acid Sequence Based (NASBA, NucliSens EasyQ, bioMérieux)
· In house PCR, MCRZ
· Virological PCR panel.

As a reference, serological diagnosis using enzyme immunoassays will be chosen.
Comparison with the current state of the art methodology will be possible.

Patients suspected for CAP will be asked to participate. Patients will be
informed by the medical practitioner and asked for informed consent.For minors
under 18, the parent(s) or guardian(s) will be asked for informed consent.
Minors between 12 and 18 also need to sign themselves.
The risks considered with participation can be considered negligible and the
burden for obtaining a throat swab, a nasopharyngeal aspirate and a urine
sample can be considered minimal.
Because all people, including minors, can get CAP, it is necessary to include
all patients.