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Performance of Respifinder Smart 22 for fast diagnosis
of respiratory tract infections
EMMD
12-14/10/2011
RAYMAEKERS M.1*, CARTUYVELS R. 1*, DUSON G.2*, FRANS J. 2*, LAMBIN S.3*, VAN DEN
ABEELE A.3*, VANKEERBERGHEN A.4*, DIERICKX K.4* DE BEENHOUWER H.4*
* on behalf of the Bilulu Study Group
1 Jessa Ziekenhuis, campus Virga Jesse, Hasselt, 2 Imelda Ziekenhuis, Bonheiden, 3 AZ Sint-Lucas, Gent, 4 Onze-Lieve-Vrouw Ziekenhuis, Aalst
Corresponding author: Stadsomvaart 11, 3500 Hasselt, BELGIUM
Phone: +32-11 30 97 02
Fax: +32-11 30 97 50
Email: [email protected]
OBJECTIVES
More than 20 viruses have been shown to cause respiratory tract infections. Identification of the etiological agent is useful to provide – or avoid – specific therapy,
to take the necessary hospital hygiene measures and for epidemiological reasons. This might result in lower treatment and hospitalisation costs. Multi parameter
nucleic acid amplification techniques allow broad spectrum detection. This multicentre study evaluates the performance of the Respifinder Smart 22 (Pathofinder)
(Smart), a multiplex analysis detecting 16 RNA viruses, 2 DNA viruses and 4 bacteria in 2 separate reactions.
METHODS
Timeframe
40 min
200 µl
NAP eSwab
(Copan)
130 min
70 min
Reverse
Transcription
(RT) + Preamplification
Hybridization
with specific
probes for
every pathogen
145 min
Fluorescence
detection
during melting
(Rotorgene)
Ligation/
Amplification/
Melting curve
Extraction in 100 µl on
Nuclisens EasyMAG
(Biomérieux)
Figure 1: Respifinder Smart 22 workflow
A total of 157 quality control samples (QCMD and Instand) and 82 patient samples, selected from the past 4 years, were analysed. The complete workflow
from extraction till analysis of the results is demonstrated in figure 1. An internal control is added to every sample before extraction to check for inhibition and
extraction efficiency and a negative sample was added to every run to check for contamination and to allow a correct threshold setting. After analysis of the
results, positive samples show a peak at a certain temperature in the ROX or Cy5 channel. Each peak is specific for a certain respiratory pathogen.. Patient
samples and EQC samples with discordant results were reanalysed with a specific single-or duplex real-time PCR (QPCR).
RESULTS AND DISCUSSION
Of all EQC samples , 34 (22%) were false negative (Table 1). The low concentration of the pathogen in the sample (Ct ≥ 35 or “highest dilution”) could be an
explanation for 23 of these samples. Of the samples with a higher concentration (Ct < 35), 1 Adenovirus type 31 sample and 1 hMPV-A1 sample were false
negative. For Legionella pneumophila serogroup 1, 3, 6 samples were not detected (even at high concentrations of 104 cfu/ml). For Bordetella pertussis, sensitivity
was very low (20%) and a Bordetella bronchiseptica gave a false positive result for Bordetella pertussis. The Respifinder Smart 22 kit was also not able to detect
the H5N1 influenza A subtype.
Pathogen
RSV A
RSV B
hMPV
INF A
INF B
INF A H1N1
B. pertussis
M. pneumoniae
C. pneumoniae
Adenovirus
Parainfluenzae (1, 2, 3, 4)
Rhinovirus/Entero
Coronavirus (NL 63, HKU1, OC43, 229 E)
L. pneumophila
Negative samples
Samples with other pathogens
Samples with inhibition
Total
# of samples
7
6
12
20
8
7
5
4
5
15
18
13
10
3
14
6
4
157
correct
6
6
7
12
7
5
1
3
4
14
16
11
7
0
14
5
118
incorrect
1
0
5
8
1
2
4
1
1
1
2
2
3
3
0
1
35
Table 2: Respifinder Smart 22 results of EQC samples
# of samples
correct
incorrect
RSV A
21
17
4
RSV B
Pathogen
7
7
0
hMPV
13
12
1
INF A
14
12
2
INF B
8
8
0
B. pertussis
4
2
2
M. pneu
5
3
2
C. pneu
1
1
0
Adenovirus
18
14
4
PIV 1, 2, 3, 4
15
12
Rhino/Entero
18
16
2
Coronavirus
7
ND
ND
L. pneu
3
3
3
0
Bocavirus
15
13
2
Total
149
120
22
Table 2: Respifinder Smart 22 results of all patient samples, ND: not determined by Real-time PCR
149 respiratory viruses/bacteria were detected in 82 patient samples, indicating that a lot of patients carry 2 or even more respiratory pathogens at the same time.
Discordant results were found for 22 pathogens, resulting in a concordance of 84% with QPCR (Table 2). Three samples with a low Ct-value for RSV in QPCR
were false negative with the Respifinder Smart. For Bocavirus, Rhinovirus/Enterovirus and PIV2 all incorrect results were positive with Smart and negative with QPCR, indicating that Smart may be more sensitive than the used Q-PCR method. For Adenovirus, all 4 incorrect samples were false negative, but they had a high
Ct value with Q-PCR. These 4 samples also contained another respiratory pathogen with a much higher viral load, indicating that Adenovirus is probably not the
causative agent of the patients disease.
CONCLUSIONS
 Respifinder Smart 22 provides reliable results for most of the respiratory pathogens detected by the kit.
 The kit is not sensitive enough for Bordetella pertussis and Legionella pneumophila
 Influenza A H5N1 and some strains of RSV are not detected by the Respifinder Smart 22 kit