Download Molecular diagnosis of bloodstream infections with a new dual

Journal of Medical Microbiology (2013), 62, 1673–1679
DOI 10.1099/jmm.0.064758-0
Molecular diagnosis of bloodstream infections with
a new dual-priming oligonucleotide-based multiplex
PCR assay
Lucrecia Carrara,1,2 Ferran Navarro,1,2 Miquel Turbau,2,3 Montse Seres,2,3
Indalecio Morán,2,4 Isabel Quintana,2,4 Rodrigo Martino,2,5
Yesica González,1,2 Albert Brell,1,2 Oscar Cordon,1,2 Karol Diestra,1,2
Caterina Mata,1,2 Beatriz Mirelis1,2 and Pere Coll1,2
Correspondence
1
Ferran Navarro
2
Servicio de Microbiologı́a, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain
Biomedical Research Institute Sant Pau (IIB Sant Pau), Sant Antoni Ma Claret 167,
08025 Barcelona, Spain
[email protected]
3
Servicio de Urgencias Generales, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain
4
Servicio de Medicina Intensiva, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain
5
Servicio de Hematologı́a, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain
Received 25 June 2013
Accepted 4 August 2013
Mortality from bloodstream infections (BSIs) correlates with diagnostic delay and the use of
inappropriate empirical treatment. Early PCR-based diagnosis could decrease inappropriate
treatment, improving patient outcome. The aim of the present study was to assess the clinical
utility of this molecular technology to diagnose BSIs. We assessed a new dual-priming
oligonucleotide-based multiplex PCR assay, the Magicplex Sepsis Test (MST) (Seegene), along
with blood culture (BC). A total of 267 patients from the intensive care unit and haematology and
emergency departments were enrolled. Clinical data were also used by physicians to determine
the likelihood of infection. Ninety-eight (37 %) specimens were positive: 29 (11 %) by both the
MST and BC, 29 (11 %) by the MST only, and 40 (15 %) by BC only. The proportion of agreement
between the two methods was 73 % (Cohen’s k: 0.45; 0.28–0.6; indicating fair to moderate
agreement). According to clinical assessment, 63 (64 %) positive specimens were considered
BSIs: 23 (36 %) were positive by both the MST and BC, 22 (35 %) were positive only by BC, and
18 (29 %) were positive only by the MST. Thirty-eight (14 %) positive specimens by the MST and/
or BC were considered as contaminants. Of 101 specimens collected from patients receiving
antibiotics, 20 (20 %) were positive by the MST and 32 (32 %) by BC. Sensitivity and specificity
were 65 % and 92 %, respectively, for the MST and 71 % and 88 %, respectively for BC. We
concluded that the MST shows a high specificity but changes in design are needed to increase
bacteraemia detection. For viability in clinical laboratories, technical improvements are also
required to further automate the process.
INTRODUCTION
Bloodstream infection (BSI) is a serious condition that
causes around 215 000 deaths per year in the USA and
135 000 in Europe (Bassetti et al., 2012; Mancini et al.,
2010; Wolk & Fiorello, 2010a). A significant cause of this
high mortality is the delay in microbiological diagnosis
Abbreviations: BC, blood culture; BSI, bloodstream infection; CoNS,
coagulase-negative Staphylococcus; DPO, dual-priming oligonucleotide;
MST, Magicplex Sepsis Test; READ, real amplicon detection; TTP, time
to positivity.
Supplementary material is available with the online version of this paper.
064758 G 2013 SGM
(Mancini et al., 2010; Schaub et al., 2011; Wolk & Fiorello,
2010b). Standard diagnosis, i.e. blood culture (BC), takes
up to 5 days – a time lapse that forces physicians to use
empirical antibiotic treatment. Kumar et al. (2006) showed
that mortality from BSI increases about 7–8 % per hour of
delay in effective antimicrobial administration within the
first hour of documented hypotension in adult patients
with septic shock.
In parallel, several studies have shown that the earlier
specific treatment is established, the better the outcome of
patients (Dellinger et al., 2008; Rivers et al., 2012). All these
studies suggest that diagnostic methods should be
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1673
L. Carrara and others
improved (Mancini et al., 2010; Paolucci et al., 2010;
Schaub et al., 2011; Wolk & Fiorello, 2010b). The key to
improving management of BSI is to reduce the time taken
to obtain microbiological results.
The concept of theranostics could be introduced into the
medical management of BSI. Theranostics in infectious
diseases implies using rapid and accurate diagnostic assays
to enable better initial management of patients and more
efficient use of antimicrobials (Picard & Bergeron, 2002).
Multiplex PCR assays are promising approaches that aim
to reduce aetiological diagnosis of BSI to hours (Paolucci
et al., 2010; Reinhart et al., 2012), avoiding the need for
culture-based microbiological methods. Several molecular
assays (PCR/ES-MS, SeptiTest, SeptiFast, Vyoo/Looxter)
shorten the time of detection of many micro-organisms to
6–8 h, but some of them do not detect drug resistance
genes (Bloos et al., 2012; Ecker et al., 2010; Fitting et al.,
2012; Lucignano et al., 2011; Tsalik et al., 2010; Wallet et al.,
2010; Wellinghausen et al., 2009). To date, neither BC nor
molecular approaches are as powerful as we would like
(Baron, 2006).
A new commercial kit, the Magicplex Sepsis Test (MST;
Seegene) appeared on the market in July 2010. The MST
was designed to detect most pathogenic micro-organisms
responsible for BSIs as well as three key drug resistance
genes, mecA, vanA and vanB, within 6 h. The assay uses a
selective human cell lysis pre-treatment of specimens to
increase detection of bacterial nucleic acids. A dualpriming oligonucleotide (DPO) is then used for amplification. DPO consists of two functional priming regions
separated by a poly(I) linker. It generates dual-priming
regions, resulting in only target-specific products (Chun
et al., 2007). Moreover, DPO primers are labelled with a
fluorescence marker, which allows early detection of
fluorescence signals [READ (real amplicon detection)
technology] and gives quicker results.
The aim of the present study was to assess the clinical
utility of this new technology to diagnose BSIs.
METHODS
This prospective study was performed at Hospital de la Santa Creu i
Sant Pau – a tertiary university hospital in Barcelona. Patients were
recruited between April 2011 and September 2011 from the intensive
care unit (ICU) and haematology and emergency departments.
Demographic data of patients, clinical and laboratory findings,
cultures of suspected source of infection, and administration of
antimicrobial therapy were recorded prospectively to determine
patients’ infection status. One blood specimen per patient and
episode was selected for the MST on the basis of suspected sepsis.
Blood samples were selected regardless of previous antibiotic
treatment. Patients younger than 18 years were excluded from the
study. During weekends, samples for the molecular approach were
not processed and they were therefore excluded from the study. The
study was approved by the Ethics Committee at Hospital de la Santa
Creu i Sant Pau (study no. 11BSP-MOL-2011-62).
BC processing. Blood specimens were collected by direct venipunc-
ture. In 11 cases, venipuncture was not available for sampling and
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specimens were obtained from catheters. About 10–20 ml of blood
was inoculated in each BC bottle (aerobic and anaerobic culture) and
incubated as soon as possible at 35 uC in BacT/Alert (bioMérieux) BC
cabinets for 7 days. When growth in BC bottles was automatically
detected, Gram stain and subcultures were performed, and isolates
were identified according to standard laboratory methods (Baron,
2005). Antibiotic susceptibility was determined using accredited
routine laboratory methods (CLSI, 2012). Genotypic identification by
16S rDNA sequencing was required in two anaerobe isolates and was
performed as previously described (http://rdna4.ridom.de/static/
primer.html; Esparcia et al., 2011; Petti, 2007).
MST processing. An aliquot of 1 ml of fresh whole blood was
required for the MST test, according to the manufacturer’s
instructions. The paired samples used for BC and the MST derived
from the same venipuncture or catheter draw. Specimens were
transported in EDTA tubes to the laboratory and stored at 2–8 uC
until processing. Specimens were discarded if the MST was not
performed within 24 h. Microbial nucleic acids were enriched by
performing a pre-treatment with a Blood Pathogen kit (Seegene)
according to the manufacturer’s instructions. This kit allows selective
human cell lysis and degradation of the released (human and nonhuman) DNA by a DNase. Intact microbial cells (viable and nonviable) were then concentrated from the lysed blood (Wellinghausen
et al., 2009). Microbial nucleic acids were automatically extracted by
the SEEPREP12 purification system (Seegene) according to the
manufacturer’s instructions. The extraction procedure took approximately 2.5 h.
Nucleic acids were amplified using a conventional PCR thermocycler
(System 9700; Applied Biosystems) in two independent reaction
tubes: one for Gram-negative bacteria and fungi, and another for
Gram-positive bacteria and three drug resistance markers: mecA,
vanA and vanB. This initial amplification process took approximately
2.5 h (Fig. 1a). Amplicon specimens were then detected, screened and
identified using SmartCycler II real-time PCR (Cepheid). Screening
and identification steps were performed separately. For the screening
step, three real-time PCRs were performed (approximately 30 min):
one for Gram-positive bacteria, one for drug resistance markers, and
one for Gram-negative bacteria and fungal pathogens (Fig. 1b). If
amplification occurred, the pathogen was identified using suitable
identification probes (Fig. 1c), taking an additional 30 min.
Amplicons were detected by fluorescent nucleotide probes and results
were analysed by Seegene VIEWER real-time PCR software. An internal
control was used in all amplification steps. For the staphylococcal
amplification probe, a Ct of ,10 cycles was needed to determine
positivity according to the manufacturer’s instructions. For the other
probes, Ct had to be ,15 cycles. To minimize contamination, DNA
extraction and amplification/detection were performed in separate
rooms. The MST was performed by specially trained laboratory
technicians that had been working with molecular techniques (both
manual and automatic) for many years.
Analyses of microbiological results. To determine whether the
micro-organisms corresponded to a true pathogen, we used recommended microbiological criteria as previously described (Weinstein &
Doern, 2011). Accordingly, the following data were taken into
consideration: identification and antimicrobial resistance pattern of
micro-organisms, and clinical findings in the patient (e.g. fever,
leukocytosis, imaging studies). Possible contaminant micro-organisms
detected only by the MST were considered true pathogens when clinical
data and additional clinical cultures supported this consideration. It
should be pointed out that antibiotic susceptibility patterns cannot be
used with the MST to compare isolates from different specimens.
Clinicians were not informed about the MST results until the end of
the study. These results did not therefore influence the medical
management of patients.
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Journal of Medical Microbiology 62
Molecular diagnosis of sepsis
(a)
Amplification
Amplicon Bank 1. Gram-positive bacteria/drug resistance
73 Gram-positive bacteria, three drug resistance markers
Amplicon Bank 2. Gram-negative bacteria/fungi
12 Gram-negative bacteria, six fungi
(b)
(c)
Screening
Gram-positive bacteria screening
Streptococcus spp.
Enterococcus spp.
Staphylococcus spp.
Drug resistance screening
vanA
vanB
mecA
Gram-negative bacteria/fungal screening
Gram-negative bacteria-A
Gram-negative bacteria-B
Fungi
Identification
Enterococcus spp.
ID 1. Streptococcus spp.
E. faecalis
S. agalactiae
S. pyogenes
E. gallinarum
S. pneumoniae
E. faecium
ID 3. Staphylococcus spp.
S. epidermidis
S. haemolyticus
S. aureus
ID 4. Gram-negative bacteria-A ID 5. Gram-negative bacteria-A
Pseudomonas aeruginosa
Serratia marcescens
Acinetobacter baumannii
Bacteroides fragilis
Stenotrophomonas maltophilia
Salmonella typhi
ID 6. Gram-negative bacteria-B ID 7. Gram-negative bacteria-B
Klebsiella pneumoniae
Escherichia coli
Klebsiella oxytoca
Enterobacter cloacae
Proteus mirabilis
Enterobacter aerogenes
ID 8. Fungi
ID 9. Fungi
Candida albicans
Candida glabrata
Candida tropicalis
Candida krusei
Aspergillus fumigatus
Candida parapsilosis
Fig. 1. Amplification (a), screening (b) and identification (c) panels of the MST.
Vassar Stats website (http://vassarstats.net/). In all cases, the 95 %
confidence intervals are shown. Cohen’s k values of 0.41–0.60 are
considered of moderate agreement and values of 0.21–0.40 are
considered of fair agreement.
In total, 101 of 267 specimens were collected from patients
receiving antibiotic treatment: 20 specimens (20 %; 13–
29 %) were positive by the MST and 32 specimens (32 %;
23–41 %) were positive by BC. Six specimens (6 %; 2–
13 %) were positive only by the MST.
RESULTS
Results of the MST and BC according to clinical
assessment (Table 2)
Statistical analyses. Statistical analyses were performed using the
From the 267 paired MST and BC set specimens, 142 came
from the emergency department, 99 from ICU areas and 26
from the haematology department. Specimens were collected
by venipuncture in 256 cases and by catheter in 11 cases.
Global results of the MST and BC (Table 1)
Ninety-eight of 267 (37 %; 31–43 %) specimens were positive:
29 (11 %; 8–15 %) by both the MST and BC, 29 (11 %; 8–
15 %) by the MST only, and 40 (15 %; 11–20 %) by BC only.
The percentage of agreement between the two methods was
73 % (Cohen’s k: 0.45; 0.28–0.6; indicating fair to moderate
agreement). Thirty-eight of 98 (14 % of 267; 11–19 %) positive
specimens were considered as contaminants: three (1 %; 0.4–
3 %) by both the MST and BC, 14 (5 %; 3–9 %) by the MST
only, and 21 (8 %; 5–12 %) by BC only. In specimens
considered as contaminant, time to positivity (TTP) for BC
was 31 h (20–72 h) and the Ct of the MST was .13.
http://jmm.sgmjournals.org
Sixty-three of 98 (64 %; 54–73 %) positive specimens (by
the MST and/or BC) were considered BSIs. Global
sensitivity and specificity of the MST for BSI detection
were 65 % (52–76 %) and 92 % (87–95 %), respectively,
whereas for BC they were 71 % (58–82 %) and 88 % (83–
92 %), respectively. In specimens considered as BSIs, TTP
for BC was 15 h (8–39 h) and the Ct of the MST was ,13.
Among specimens obtained from catheters, only three were
positive by the MST (two S. epidermidis and one K.
pneumoniae). Both S. epidermidis specimens were considered true bacteraemia according to other specimens
(including the paired BC) and clinical data.
MST-positive specimens. Forty-one of the 58 MST-
positive specimens were considered as BSIs: 23 (36 %; 25–
50 %) specimens were positive by both the MST and
BC (MST-positive/BC-positive) and 18 (29 %; 18–42 %)
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L. Carrara and others
Table 1. Global results of the MST and BC
Result
MST-positive
MST-negative
Total
BC-positive
BC-negative
Total
29*Dd§
40
69
29||
169
198
58
209
267
*BC detected two additional Bacteroides fragilis and coagulasenegative Staphylococcus (CoNS) in two different specimens.
DMST detected two additional Staphylococcus epidermidis and
Streptococcus agalactiae in two different specimens.
dIn three specimens the MST was positive for: Klebsiella pneumoniae
in two cases and for Pseudomonas aeruginosa in the other while CoNS
isolated by BC were considered as contaminants.
§Three positive specimens by both the MST and BC were considered
as contaminants.
||Ten were Escherichia coli (two by polymicrobial detection: K.
pneumoniae and E. coli), two K. pneumoniae, two Streptococcus
pneumoniae, one Enterobacter cloacae. The remaining specimens were
considered as contaminants.
Six were E. coli, three CoNS, two P. aeruginosa, two by
polymicrobial detection: P. aeruginosa and CoNS, one Klebsiella
oxytoca, one K. pneumoniae, one S. pneumoniae, two Streptococcus
dysgalactiae subsp. equisimilis, one Atopobium rimae, one Bacillus
spp., one Corynebacterium spp., one Odoribacter splanchnicus (see
supplementary material available with the online version of this
paper). The remaining positive specimens were considered as
contaminants.
specimens were positive only by the MST (MST-positive/
BC-negative). Among these specimens, 13 (88.9 %; 67.2–
96.9 %) were MST-positive for Enterobacteriaceae (two of
these samples were positive for both K. pneumonia and E.
coli). Seven specimens were collected from patients under
antibiotic treatment (Tables 1, 2 and 3).
BC-positive specimens. Forty-five of 69 BC positive
specimens were considered as BSIs: 22 (35 %; 24–48 %)
specimens were positive only by BC (MST-negative/BCpositive). Four specimens were positive for P. aeruginosa;
MST amplification was performed on the isolates, giving a
positive result for P. aeruginosa. The MST was inhibited in
one specimen that was BC-positive for Staphylococcus
haemolyticus. In four specimens, BCs were positive by
species not included in MST detection and identification
panels. The remaining 13 specimens are detailed in Tables
1, 2 and 3. We found no statistical significance between
the number of pathogens detected by the two methods
(63) and the number of pathogens (45) detected by BC
alone (P.0.05).
Regarding drug resistance markers, 10 meticillin-resistant
staphylococci were detected by antibiotic susceptibility test
(eight CoNS and two S. aureus). The MST detected the
mecA gene in four of these specimens (three CoNS and one
S. aureus). In the remaining six specimens, three MSTs
were negative, two MSTs were positive only for
Staphylococcus spp. and failed to detect both the species
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and mecA gene, and one MST was inhibited (no Ct detected
for internal control). The Ct of the identification probe of
one of the meticillin-resistant S. aureus was 10.02. As the Ct
for Staphylococcus spp. is 10, the software scored this
specimen as negative.
In two specimens, the MST detected the mecA gene but
failed to detect any organism. Vancomycin-resistant
enterococci were not detected in the present study either
by the MST or by the antibiotic susceptibility test.
DISCUSSION
This is the first study to assess the clinical performance of
the multiplex PCR assay, the Magicplex Sepsis Test. We
found that sensitivity for the MST (65 %) was not higher
than for BC (71 %) but was comparable to the rates found
in similar studies (52–87 %) using other commercial kits
(Bloos et al., 2012; Fitting et al., 2012; Tsalik et al., 2010;
Wallet et al., 2010). However, a remarkable proportion
(37 %) of Enterobacteriaceae spp. was detected by the MST
but not by BC. BC detected a higher number of P.
aeruginosa isolates than the MST (four of five). Only one of
267 MST amplifications was inhibited. The proportion of
agreement between the MST and BC was 73 %, mostly due
to negative results. The ability of the MST to detect whole
cells, viable or not, should increase the number of positive
specimens processed under the antimicrobial effect.
Unexpectedly, the number of positive specimens processed
under the antimicrobial effect was not higher by the MST
than by BC. The most striking finding was the discrepancy
between positive MST and BC results. We detected more
discrepant results than in previous studies (Bloos et al.,
2012; Tsalik et al., 2010; Wallet et al., 2010).
Explanations suggested for PCR-negative/BC-positive
results are genetic variability or mutations of the target
site, inhibition of PCR and low microbial load (Bloos et al.,
2012; Esparcia et al., 2011; Wellinghausen et al., 2009). Our
current findings do not fully confirm earlier works.
Mutations of the target alteration were excluded for P.
aeruginosa because MST amplification performed in the
isolates was positive. Only one MST amplification was
inhibited (1/267; 0.4 %). The bacterial load was indirectly
measured by the TTP. The mean TTP for all MST-positive/
BC-positive results was 15 h. Nevertheless, the mean TTP
for all MST-negative/BC-positive results was 19 h.
Accordingly, the hypothesis of a low bacterial load can be
ruled out. In this study, the blood volume used for the
MST was smaller than in other studies (Bloos et al., 2012;
Fitting et al., 2012; Tsalik et al., 2010; Wallet et al., 2010).
The significant difference in the sample volumes used for
the MST and BC testing is likely a major contributing
factor to the poor sensitivity of PCR. Hansen et al. (2009)
evaluated different pre-analysis sample treatments together
with various DNA extraction kits for the selective isolation
of bacterial DNA from whole blood. They concluded that
the combination of performing a pre-analysis sample
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Molecular diagnosis of sepsis
Table 2. Results of the MST and BC compared to clinical
criteria
Results
BSI
BSI
MST
Positive
Negative
Total
BC
Positive
Negative
Total
Not BSI
Total
41
22
63
17*
187
204
58
209
267
45
18
63
24*
180
204
69
198
267
MST values: sensitivity 65 % (52–76 %), specificity 92 % (87–95 %),
positive predictive value 71 % (57–82 %), negative predictive value
89 % (84–93 %), Cohen’s unweighted k50.45.
BC values: sensitivity 71 % (58–82 %), specificity 88 % (83–92 %),
positive predictive value 68 % (55 279 %), negative predictive value
91 % (86–94 %), Cohen’s unweighted k50.45.
*Non-proven BSIs with positive specimens either by BC or the MST
were considered as contaminated specimens.
treatment and using a larger sample volume increased the
bacterial detection limit (Hansen et al., 2009). Accordingly,
in our molecular approach, the volume of blood requested
should probably be increased to improve sensitivity.
A possible explanation for the PCR-positive/BC-negative
results could be the use of antimicrobial agents before blood
extraction (Wellinghausen et al., 2009). These agents affect
the viability of micro-organisms, diminishing the ability of
BC to detect bacteraemia. Nevertheless, there were no
significant differences between percentages of positivity
by BC and the MST (32 % and 20 %, respectively) in the
specimens collected from patients receiving antibiotic
treatment. These results do not support the notion that
micro-organisms detected by PCR may have been nonviable and thus non-detectable by BC (Hansen et al., 2009;
Wellinghausen et al., 2009). Moreover, to give PCR-positive/
BC-negative results could be misleading to the clinician and
cause an unnecessary change in antimicrobial therapy. These
discrepancies could be explained by an unmeasured
combination of bacterial load, bacterial viability and
decreased antimicrobial concentration in BC.
The MST failed to detect five staphylococcal BSIs (one S.
aureus). The manufacturer has proposed a cut-off of 10
to diminish the number of Staphylococcus spp. falsepositive results. However, some bacteraemia caused by
Staphylococcus spp. could be missed (one in our series). A
Ct cut-off of 15 as for the other micro-organisms could be
better to detect staphylococcal BSIs.
Despite its low sensitivity (65 %), we would like to emphasize
that the MST is a reliable tool to detect bacteraemia,
particularly those produced by Enterobacteriaceae. A DPO
design, along with the pre-treatment of samples, reduced
both false-negative and false-positive results (Kommedal
et al., 2012). The MST could therefore be useful and reliable
for the selection of an antibiotic, even in those meticillinresistant Staphylococcus in which the MST detected a mecA
Table 3. Total micro-organisms detected by the MST and BC
Species
Enterobacter cloacae
Enterococcus faecalis
Escherichia coli
Klebsiella oxytoca
Klebsiella pneumoniae
Pseudomonas aeruginosa
Proteus mirabilis
CoNS
Streptococcus pneumoniae
Staphylococcus aureus oxaS
Staphylococcus aureus oxaR
Streptococcus dysgalactiae
Streptococcus viridans
Bacillus spp.
Corynebacterium spp.
Anaerobe
Candida spp.
Totally detected
Detected by
the MST
Detected by
BC
MST
control
BC
control
2
2
20
1
11
5
1
44
4
3
2
3
1
1
4
4
1
2
1*
13
0
10
1
1
21
3
3
1
1D
0
0
0
0
0
0
2
10
1
5
4
1
30
2
3
2
3
1
1
4
4
1
–
0
0
0
0
0
0
16
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
20
0
0
0
0
1
1
3
0
0
Coincidence (%)
0/2
1/2
4/20
0/1
4/11
0/5
1/1
7/44
1/4
3/3
1/2
1/3
0/1
0/1
0/4
0/4
0/1
(0)
(50)
(20)
(0)
(36)
(0)
(100)
(16)
(25)
(100)
(50)
(30)
(0)
(0)
(0)
(0)
(0)
*MST detected Enterococcus sp.
DMST detected Streptococcus sp.
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L. Carrara and others
resistance gene. This should make the MST a practical kit for
clinical laboratories.
The MST is also a reliable tool to detect bacteraemia within
6 h. Its DPO design makes it an attractive primer set in this
setting, where false-negative and false-positive PCRs can
be reduced by preventing human DNA cross-reactivity
(Kommedal et al., 2012). We emphasize here that the MST
allowed us to detect Enterobacteriaceae that were not
detected in BC. Some important technological features
could be improved, however.
In our experience, the MST technology is hard to use as
a diagnostic tool in clinical microbiology laboratories
that provide service 24/7. The whole process requires
four manual steps of manipulation: pre-treatment of
specimens, extraction of DNA, screening amplification
and amplification to identify the micro-organism. The
use of the conventional PCR is a handicap for the
MST because of potential contamination of PCR
reagents by amplicons. Technological features should
thus be modified accordingly. We suggest increasing
the volume of the specimens for the MST and homogenizing the cut-off for all micro-organisms, including
Staphylococcus. Both these changes would help to
increase the detection of bacteraemia. Finally, the whole
procedure of the MST should be automated, as this
would facilitate the integration of the MST in microbiology laboratories.
A positive point of the MST is its ability to detect resistance
genes such as mecA and van. Nevertheless, in our
experience, the MST failed to detect six out of 10
meticillin-resistant genes. Moreover, the detection of
additional resistance genes such as those involved in
carbapenem resistance, e.g. blaKPC , blaVIM, blaIMP, and
blaOXA-48, would be beneficial. This point is particularly
relevant for countries where carbapenem resistance is even
more frequent than vancomycin resistance. The MST could
therefore be useful for the selection of an antibiotic within
the first hours of BSI.
In conclusion, the MST shows high specificity but its rate
of positivity is similar to that of BC. Changes in design are
needed to increase bacteraemia detection and technical
improvements are needed to further automate the process
for viability in clinical laboratories.
Baron, E. J. (editor) (2005). Cumitech 1 C: Blood Cultures IV.
Washington, DC: American Society for Microbiology.
Baron, E. J. (2006). Implications of new technology for infectious
diseases practice. Clin Infect Dis 43, 1318–1323.
Bassetti, M., Trecarichi, E. M., Mesini, A., Spanu, T., Giacobbe, D. R.,
Rossi, M., Shenone, E., Pascale, G. D., Molinari, M. P. & other
authors (2012). Risk factors and mortality of healthcare-associated
and community-acquired Staphylococcus aureus bacteraemia. Clin
Microbiol Infect 18, 862–869.
Bloos, F., Sachse, S., Kortgen, A., Pletz, M. W., Lehmann, M.,
Straube, E., Riedemann, N. C., Reinhart, K. & Bauer, M. (2012).
Evaluation of a polymerase chain reaction assay for pathogen
detection in septic patients under routine condition: an observational
study. PLoS ONE 7, e46003.
Chun, J. Y., Kim, K. J., Hwang, I. T., Kim, Y. J., Lee, D. H., Lee, I. K. &
Kim, J. K. (2007). Dual priming oligonucleotide system for the
multiplex detection of respiratory viruses and SNP genotyping of
CYP2C19 gene. Nucleic Acids Res 35, e40.
CLSI (2012). Performance Standards for Antimicrobial Susceptibility
Testing; 22nd International Supplement M100–S22. Wayne, PA:
Clinical and Laboratory Standards Institute.
Dellinger, R. P., Levy, M. M., Carlet, J. M., Bion, J., Parker, M. M.,
Jaeschke, R., Reinhart, K., Angus, D. C., Brun-Buisson, C. & other
authors (2008). Surviving sepsis campaign: international guidelines
for management of severe sepsis and septic shock: 2008. Crit Care
Med 36, 296–327.
Ecker, D. J., Sampath, R., Li, H., Massire, C., Matthews, H. E., Toleno,
D., Hall, T. A., Blyn, L. B., Eshoo, M. W. & other authors (2010). New
technology for rapid molecular diagnosis of bloodstream infections.
Expert Rev Mol Diagn 10, 399–415.
Esparcia, O., Montemayor, M., Ginovart, G., Pomar, V., Soriano, G.,
Pericas, R., Gurgui, M., Sulleiro, E., Prats, G. & other authors (2011).
Diagnostic accuracy of a 16S ribosomal DNA gene-based molecular
technique (RT-PCR, microarray, and sequencing) for bacterial
meningitis, early-onset neonatal sepsis, and spontaneous bacterial
peritonitis. Diagn Microbiol Infect Dis 69, 153–160.
Fitting, C., Parlato, M., Adib-Conquy, M., Memain, N., Philippart, F.,
Misset, B., Monchi, M., Cavaillon, J. M. & Adrie, C. (2012). DNAemia
detection by multiplex PCR and biomarkers for infection in systemic
inflammatory response syndrome patients. PLoS ONE 7, e38916.
Hansen, W. L., Bruggeman, C. A. & Wolffs, P. F. (2009). Evaluation of
new preanalysis sample treatment tools and DNA isolation protocols
to improve bacterial pathogen detection in whole blood. J Clin
Microbiol 47, 2629–2631.
Kommedal, O., Simmon, K., Karaca, D., Langeland, N. & Wiker, H. G.
(2012). Dual priming oligonucleotides for broad-range amplification
of the bacterial 16S rRNA gene directly from human clinical
specimens. J Clin Microbiol 50, 1289–1294.
Kumar, A., Roberts, D., Wood, K. E., Light, B., Parrillo, J. E., Sharma,
S., Suppes, R., Feinstein, D., Zanotti, S. & other authors (2006).
ACKNOWLEDGEMENTS
This study was partially supported by the Ministy of Health and
Consumer Affairs, Instituto de Salud Carlos III-FEDER, Spanish
Network for Research in Infectious Diseases (REIPI/RD06/0008/
0013). We thank the IZASA-Werfen Group for providing diagnostic
kits and C. Newey for revising the English. All authors except K. D.
and C. M. declare that they have no conflict of interest. Both K. D.
and C. M. are now working with the IZASA-Werfen Group. Results of
this study were presented at the XVI Congress of the Spanish Society
of Microbiology and Infectious Diseases, 9–11 May 2012, Bilbao,
Spain.
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REFERENCES
Duration of hypotension before initiation of effective antimicrobial
therapy is the critical determinant of survival in human septic shock.
Crit Care Med 34, 1589–1596.
Lucignano, B., Ranno, S., Liesenfeld, O., Pizzorno, B., Putignani, L.,
Bernaschi, P. & Menichella, D. (2011). Multiplex PCR allows rapid
and accurate diagnosis of bloodstream infections in newborns and
children with suspected sepsis. J Clin Microbiol 49, 2252–2258.
Mancini, N., Carletti, S., Ghidoli, N., Cichero, P., Burioni, R. &
Clementi, M. (2010). The era of molecular and other non-culture-
based methods in diagnosis of sepsis. Clin Microbiol Rev 23, 235–251.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 04 May 2017 03:44:02
Journal of Medical Microbiology 62
Molecular diagnosis of sepsis
Paolucci, M., Landini, M. P. & Sambri, V. (2010). Conventional and
molecular techniques for the early diagnosis of bacteraemia. Int J
Antimicrob Agents 36 (Suppl 2), S6–S16.
Petti, C. A. (2007). Detection and identification of microorganisms by
gene amplification and sequencing. Clin Infect Dis 44, 1108–1114.
Picard, F. J. & Bergeron, M. G. (2002). Rapid molecular theranostics
in infectious diseases. Drug Discov Today 7, 1092–1101.
admitted from the emergency department with sepsis. J Clin Microbiol
48, 26–33.
Wallet, F., Nseir, S., Baumann, L., Herwegh, S., Sendid, B., Boulo, M.,
Roussel-Delvallez, M., Durocher, A. V. & Courcol, R. J. (2010).
Preliminary clinical study using a multiplex real-time PCR test for
the detection of bacterial and fungal DNA directly in blood. Clin
Microbiol Infect 16, 774–779.
New approaches to sepsis: molecular diagnostics and biomarkers. Clin
Microbiol Rev 25, 609–634.
Weinstein, M. & Doern, G. (2011). A critical appraisal of the role of
the clinical microbiology laboratory in the diagnosis of bloodstream
infections. J Clin Microbiol 49 (9 Suppl), S26–S29.
Rivers, E. P., Katranji, M., Jaehne, K. A., Brown, S., Abou Dagher, G.,
Cannon, C. & Coba, V. (2012). Early interventions in severe sepsis and
Wellinghausen, N., Kochem, A. J., Disqué, C., Mühl, H., Gebert, S.,
Winter, J., Matten, J. & Sakka, S. G. (2009). Diagnosis of bacteremia in
septic shock: a review of the evidence one decade later. Minerva
Anestesiol 78, 712–724.
whole-blood samples by use of a commercial universal 16S rRNA genebased PCR and sequence analysis. J Clin Microbiol 47, 2759–2765.
Schaub, N., Frei, R. & Müller, C. (2011). Addressing unmet clinical
Wolk, D. & Fiorello, A. B. (2010a). Code sepsis: rapid methods to
needs in the early diagnosis of sepsis. Swiss Med Wkly 141,
w13244.
diagnose sepsis and detect hematopathogens: Part I: the impact and
attributes of sepsis. Clin Microbiol Newslett 32, 33–37.
Tsalik, E. L., Jones, D., Nicholson, B., Waring, L., Liesenfeld, O., Park,
L. P., Glickman, S. W., Caram, L. B., Langley, R. J. & other authors
(2010). Multiplex PCR to diagnose bloodstream infections in patients
Wolk, D. & Fiorello, A. B. (2010b). Code sepsis: rapid methods to
Reinhart, K., Bauer, M., Riedemann, N. C. & Hartog, C. S. (2012).
http://jmm.sgmjournals.org
diagnose sepsis and detect hematopathogens: Part II: challenges to the
laboratory diagnosis of sepsis. Clin Microbiol Newslett 32, 41–49.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 04 May 2017 03:44:02
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