Diagnosis of respiratory tract infections by molecular techniques in pediatric patients: Significance of positive samples with this ultra sensitive technique and analysis of the best sampeling method

Acute respiratory infections are one of the most important reasons for
hospitalisation of young children. This is not only a cause for morbidity and
mortality of the individual, but can also be a reason for healthcare cost due
to hospitalisation, diagnostic procedures and therapy.
In a lot of cases a causing pathogen is not found, due to lack of sensitivity
of culture and serology, and because sputum can not always be obtained. This
may result in suboptimal treatment and longer hospitalisation. More sensitive
and rapid testing is needed to increase pathogen detection rates so adequate
treatment can be given.

The objective of the study is to get more insight into the interpretation of
multiplex PCR test (which are so sensitive, that falls positive samples due to
colonisation, or interpretation problems due to mixed infection may occur) and
to expand a real-time multiplex pcr assay from 14 to 20 different pathogens,
for fast and ultra sensitive detection of most important respiratory bacteria
and viruses. These pathogens are: adeno-, influenza A en B, entero, hMP-, RS-,
rhino-, corona-, parecho- en parainfluenza- 1,2,3,4, boca- virus,
Streptococcus pneumnoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae,
Chlamydophila psitacci, Legionella pneumophila en Bordetella pertussis en
parapertussis.

Key questions are:
- What is the frequency of the different pathogens when molecular test are used.
- Are there associations with clinical features?
- What is the best way of sampling for molecular tests?
- Is there a correlation between the amount (= load) of a pathogen and the
clinical picture.
- What is the meaning of multiple postive test, for different
pathogens, with this sensitive test.
- How long can DNA/RNA be detected after the onset of symptoms, with and
without treatment.

This study will be carried out in two different hospitals.
Parents of children with respiratory complaints (indoor and outdoor patients)
will be asked to participate in the study. After informed consent, 5 materials
will be taken from the patient: 1) nasopharyngeal wash, nasopharyngeal swab,
pharynx swab, a urine sample and 5ml of venous blood. If patients are still in
the hospital on day 3 and 7, nasopharyngeal wash, nasopharyngeal swab and
pharynx swab will be taken for a second and third time. Nurses specialised in
children care will perform the sampling procedures. The treating physician
shall ask for informed consent and has to fill in the case record form. The
head investigator will visit the wards and help when there are questions or
difficulties.
All data will be stored anonymously in a database and will be analysed
later.Analysed PCR results will be compared with the full spectrum of routine
techniques. Sample methods will be compared with each other.
This study will have a total duration of 2 years to include 300 patients

The nature and extent of the burden will exist of:

1) An unpleasant sensation during the sampling of nasopharyngeal samples.
2) The pharynx sample may also give a heave reflex.
3) Urine sampling with urine bag sticked to skin can give some irritation
during removel, comparble with the removel a plaster.
4) Venous puncture gives a painful sensation during the insertion of the needle
and can lead to a haematoma.

If a patient is still hospitalised on day 3 and 7 he will, this burden will be
increased with two extra episodes of nasopharyngeal and pharynx samplimg. The
total time of burden will maximally be 3x7 = 21 minutes, plus 20 minutes of
explanation
Besides these significant, but reversible and short lasting burdens, this study
does not impose a real risk on the partcipating patients.