Analytical and Clinical Validation of the 16S Gene and Antimicrobial

Resistance Genes by Real Time PCR in Blood Cultures of Patients Undergoing

Hematopoietic Stem Cells Transplant

L. C. Menezes
1
*, T. T. Rocchettis
1
, K. D. Bauab
1
, P. Cappellanos
1
, F. Carlesses
2
, J. S. R. de Oliveiras
1
, A. C. C. Pignatari
1

1
UNIFESP - Federal University of So Paulo Brazil
2
IOP- GRAACC - Support Group for Children and Adolescents with Cancer So Paulo - Brazil

Abstract
Conclusions
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The prevalence of the most important infectious agents guides the empiric antimicrobial after hematopoietic transplant therapy. Approximately
60% of episodes of fever in patients with neutropenia are frequently correlated with documented bloodstream infection (ICS). Bloodstream
infections are associated with high rates of morbidity and mortality, with a mortality rate ranging from 20% to 70%. The incidence of sepsis is
increasing the SENTRY Antimicrobial Surveillance Program (1997-2002) found ten bacterial species accounted for 89-92% of all isolates in
659.935 cases of sepsis reported in the USA in 2000. The ranking of the presence of these species was very similar across North America, Latin
America, and Europe (Biedenbach et al., 2004). Recently, the deaths was estimated that the most dangerous clinical manifestations of
bloodstream infectious, sepsis and shock, are the 10
th
leading cause of death in the United States, accounting for 6% of all deaths (50.37 deaths
per 100,000 individuals in the overall population) (Kung et al., 2008). Sepsis is caused by heterogeneous group of infectious etiologies. Early
diagnosis and provision of appropriate antimicrobial therapy correlate with positive clinical outcomes (Tsalik et al., 2010). Molecular amplification
techniques have been developed to identify microbial in bloodstream. Conserved regions of microbial genomes serve as targets for
amplification, such as the rRNA genes and the 16S-23S interspace. Many protocols that target individual species or multiple pathogens within a
particular clade have been developed, such as panbacterial and panfungal assays (Espy et al., 2006). PCR techiniques based on the automated
fluorescence detection of amplicons (real-time PCR) are usually more robust, less labor-intensive, and less prone to contamination than
conventional PCR techniques (Klouche et al., 2008).
The purpose of this study was the detection of Gram-positive and Gram-negative bacteria from automated blood cultures (Bactec

, Becton
Dickinson) and whole blood with the use of the multiplex real-time polymerase chain reaction (PCR) and detection of antimicrobial resistance
genes.












Bloodstream infections should be diagnosed and treated quickly, especially in immunecompromised patients. Molecular diagnoses are time-
saving and could provide an early appropriate antibiotic therapy. The aim of this study was to perform analytical and clinical validation of 16S
bacterial gene for bacteria detection and antimicrobial resistance genes from bottles of automated blood cultures and direct from collected blood
samples of patients undergoing hematopoietic stem cells transplant. Methods: 211 blood samples, 160 from Bactec system bottles and 51 from
collected blood of 45 patients submitted to hematopoietic stem cells transplant were analyzed. The validation was performed based on CLSI
documents EP-17, EP-12 and GP-10. The analysis of specificity was done by ROC curve and the sensitivity by the limit of detection (LoD)
definition. The 16S rDNA gene detection with Gram-specific probes was standardized by Taqman multiplex real-time. The detection of the
resistance genes bla
SHV
, bla
TEM
, bla
CTX-M
, bla
IMP
, bla
SPM
bla
VIM
bla
KPC
, vanA, vanB and mecA was done by the SYBR Green real-time technique.
Results: The assays were able to detect the target genes to a corresponding dilution of 15 to 1.5 CFU per reaction. The concordance between
Gram stain of the blood culture and PCR with Gram-specific probes was 51.5%. Oxacilin susceptibility and detection of mecA gene was 76.93%
concordant. For the ESL positives isolates and ESL gene tested the concordance was 66.6%. Vancomycin resistance and detection of vanA
gene was 100% concordant. Of the 2 isolates resistant to cabapenems none were positive for carbapenemase and metallo--lactamase genes.
Conclusion: This study suggests that multiplex PCR for detection of Gram-positive/Gram-negative bacteria and antimicrobial resistance genes
could be useful for rapid diagnosis of bloodstream infection in this patient setting.



A total of 211 blood samples from 160 blood BACTEC

cultures and 51 whole blood were obtained for 45 patients with suspicion of bloodstream
infections submitted to hematopoietic stem cells transplant at Hospital So Paulo, UNIFESP. All 160 blood cultures (BC) were phenotype
identification and antimicrobial sensibility by Phoenix

.

DNA extraction
The extraction of DNA from the blood cultures used phenol chloroform method (Brazol

, LGC, Brazil) and DNA from whole blood was performed

QIAamp DNA Mini Kit (Qiagen, Germany). The initial 200 L of blood cultures volume and 1,5 mL whole blood, briefly with 300 mg of glass
beads (diameter 0,3 mm; Scientific Industries) and processed in a disruptor Genie (Scientific Industries) for 10 minute to achieve cell lysis.


Real-time PCR
The detection of bacterial DNA was performed using universal primers of 16S rDNA gene . The differentiation between Gram-positive and Gram-
negative bacteria was used hybridization with Gram-specific probes by multiplex real-time Taqman (Bispo et al., 2010). TaqMan real-time PCR
reactions was performed in a 20 L reaction volume containing 10 L TaqMan Universal Master Mix 2X (Applied Biosystems, CA), 0,5 L (10
M) each primers 0,6 L (5 M) Gram-negative probe, 0,2 L (5 M) Gram-positive probe, 5,2 L sterile water and 3 L DNA template.
The resistance genes: bla
SHV
, bla
TEM
, bla
CTX
, bla
KPC
, bla
IMP
, bla
SPM
, bla
VIM
, vanA and mecA were detected using specific primers showed in Table
1. SYBR Green real-time PCR reactions was performed in a 25 L reaction volume containing 12,5 L Kit Platinum SYBR Green qPCR Super
Mix (Invitrogen, California, EUA), 0,75 L (10 M) each primers 6 L sterile water and 5 L DNA template. The hemochromatosis gene was used
for internal control (Forward 5 GATGACCAGCTGTTCGTGTTC 3, Reverse 5 CCACATCTGGCTTGAAATTCTACTG 3).
The reactions were performed on 7500 Real Time PCR System (Applied Biosystems, CA) using the following program: 2 min at 50 C, 10 min at
95 C, followed by 40 cycles of 15 s at 95 C, and 60 s at 60 C. For the SYBR Green PCR, the sample temperature was gradually increased to
95 C in order to generate dissociation curves. Fluorescence was quantified on-line and at the end point with the sequence detection system
software (version 2.0, Applied Biosystems). Threshold Cycle (Ct) values was obtained based on a threshold pre-established at 0.20.

Validation Real-time PCR
The limit of detection (LoD) and cutoff were evaluated according to CLSI documents EP-17, EP-12 and GP-10. The clinical sensitivity and
specificity were calculated in a receiver operating characteristic (ROC) curve. The specimen preparation was evalued using samples well
caractherized and control ATCC.
The negative control were obteined from 30 healthcare professionals volunteer provided by Division of Infectious Diseases, Federal University
of So Paulo/UNIFESP.
Spiked specimen were prepared by spiking target-negative K-EDTA blood, blood cultures and DNase/RNase-free milliQ water tubes from
bacterial reference strains to a density corresponding to a McFarland turbidity standard of 0,5 (1,5 x 10
8
CFU/mL) MacFarland each gene
studied. These specimen preparation were used to prepare serial 10-fold dilutions.

Table 1 List of Primers and sequence for amplification antimicrobial resistance genes.











Table 2 - Results of real-time PCR, culture identification and antimicrobial sensitivity by Phoenix for positive samples from bottles
of blood culture.


The real-time PCR identified the presence or absence of bacteria and the select at resistance genes. The real-time PCR sensitivity
experiment showed that the assay was capable of detecting the 16S rRNA target gene at 1 pg/L DNA concentration for both Gram-specific
probes. When different control strains were submitted to the SYBR Green Real-time PCR assay, differences in the Tm of the amplicons
were observed for antimicrobial resistance genes (Table 3).
A Ct cutoff value and Ct LoD for each genes showed in Table 3. The area under the ROC curve demonstrated a satisfactory performance
above 0.70 for most genes studied. The specificity was around 100% for all genes evaluated by ROC curve. The PCR efficiency was
acceptable for all genes. The sensitivity by ROC curve was above 80% for bla
KPC
and 16S rDNA Gram positive genes and below 60% for
bla
SHV
, bla
TEM
and vanB genes (Table 3).
Thirty three samples was detected by BACTEC

and identified by phenotypic methods, 23 Gram-positive and 10 Gram-negative. Twelve of

23 Gram-positive and five of 10 Gram-negative samples were concordantly positive by BC and real time PCR. (Table 2 ). Nine BC-negative
samples were positive by PCR.
Nine samples were concordant in detecting mecA gene compared with the phenotypic resistance to methicillin. The vanA gene was
detected in 4 Enterococcus spp (E. faecalis and E. faecium) and concordance was 100% between two Vancomycin resistance methods.
Only two samples of the five positive ESL real time PCR were identified by phenotypic method as ESL producers, other two samples were
identified ESL producers by phenotypic method and negative by real-time PCR.
The real time PCR assays performance was adjusted as follows: sensitivity, 65,38%; specificity, 88,05%; negative predictive value (NPV),
92,91%; positive predictive value (VPP), 51,51% when compared to phenotypic method.



The methodologies Multiplex Taqman and SYBR Green real-time PCR showed good analytical specificity when tested with bacterial
samples well characterized and controls ATCC.
The analytical sensitivity for the SYBR Green method was 100% concordant with the ATCC and control samples for the reactions of
internal control and resistance genes.
The correlation between phenotypic identification system by Phoenix

and molecular Gram-negative and Gram-positive by Multiplex PCR

Taqman real-time for the clinical samples was of 51,5%.
The results obtained should be interpreted together of clinical and other laboratory data and f this study suggest that multiplex PCR Gram-
positive and Gram-negative and detection of antimicrobial resistance genes by real-time PCR could be useful in the diagnosis of
bloodstream infection in patients undergoing hematopoietic stem cells transplant.
A large controlled study is in progress to further evaluate the clinical benefits of using the real-time PCR en bloodstream infection.






Federal University of So Paulo
So Paulo - SP, Brazil
Phone/Fax: +55 11 5081 2965
liana.carballo@unifesp.br
R: Resistant; S: Susceptible; * CLSI 2010; NA: results not available; ND: Not Detected; CoNS: coagulase negative Staphylococcus



Table 3 - Determined Ct values for LoD and cut off, Tm, efficiency, sensibility, specificity and area under curve calculated by ROC
curve for qPCR.


P1511
Results
Introduction and Purpose
Methods
Results
Conclusions
References
Patients Age
Bacterial isolated from
blood culture (Phoenix)
Bacterial
detected with
the PCR test
Antimicrobial susceptibility (Phoenix)
Resistance genes by
real time PCR
01 49
S. epidermidis GP S* Oxacilin mecA
S. epidermidis GP S Oxacilin mecA
S. epidermidis GP S Oxacilin mecA
C.freundii GP S
Cephalosporins and
Carbapenems
ND
S. epidermidis GP R* Oxacilin mecA
ND GP and GN NA NA bla
SHV

02 28
E. cloacae GN R Cephalosporins ND
E. cloacae GN R Cephalosporins ND
E. cloacae GN R Cephalosporins bla
SHV

03 34
CoNS GP R Oxacilin mecA
CoNS GP R Oxacilin mecA
CoNS GP R Oxacilin ND
04 37
P. putida ND NA NA ND
S. epidermidis GP R Oxacilin mecA
S. epidermidis GP R Oxacilin mecA
05 44
S. pneumoniae ND S Penicilin ND
S. pneumoniae GP S Penicilin ND
06 49
E. faecium GP R Glycopeptide vanA
E. faecium ND R Glycopeptide vanA
E. faecium ND R Glycopeptide vanA
07 53
BGNNF GN R
Cephalosporins and
Carbapenems
ND
Acinetobacter spp. ND R
Cephalosporins and
Carbapenems
ND
ND GN NA NA ND
08 38 K. pneumoniae ND R and S
Cephalosporins and
Carbapenems
ND
09 50
E. faecalis ND S Glycopeptide ND
E. faecalis GP S Glycopeptide ND
10 56
E. faecium GP R Glycopeptide vanA
ND GP NA NA ND
11 23
ND GN NA NA bla
CTX-M
ND GN NA NA ND
ND GP and GN NA NA bla
CTX-M
ND GP NA NA ND
12 27
S. epidermidis GP R Oxacilin mecA
S. epidermidis GP R Oxacilin mecA
S. epidermidis GP R Oxacilin mecA
13 64 ND ND NA NA bla
SHV

14 13 A. baumannii GN S Cephalosporins ND
15 01 E. coli GN S
Cephalosporins and
Carbapenems
ND
16 14 S. epidermidis GP R Oxacilin mecA
17 51
CoNS ND R Oxacilin ND
CoNS ND R Oxacilin ND
Microorganism
or Resistance gene
Cut off LoD T
m
SD T
m
Efficiency
%
Area under
curve ROC
Sensibility
(%)
Especificity
(%)
GP 16S rDNA
a
40 38,6 - - 109,1 0,995 87,5 99,97
GN 16S rDNA
b
36,6 34,4 - - 94,73 0,998 76,5 99,99
mecA 34,9 33,91 71,92 0,29 108,9 0,976 77,1 99,95
vanA 35,5 33,67 85,48 0,08 93,7 0,990 74,5 100,00
vanB 33,3 32,29 74,41 0,14 116,91 0,961 57,9 99,96
bla
KPC
37,9 36,67 91,31 0,07 81,2 0,984 82,7 99,96
bla
TEM
36,4 34,53 85,85 0,22 81,9 0,996 67,3 99,96
bla
SHV
34,1 33,26 91,67 0,22 81,4 0,863 25,7 100,00
bla
CTX
33,7 32,62 88,85 0,09 86,4 0,896 70,8 99,97
bla
IMP
33,23 28,49 76,27 0,10 100,0 0,954 67,6 100,00
bla
SPM
33,23 30,99 84,85 0,10 80,0 0,981 73,7 98,30
bla
VIM
33,23 31,82 89,43 0,10 128,7 0,938 72,2 98,30
Primer and Probe Sequence (3)
Microorganism or Antimicrobial
Resistance
Gene
GenBank Accession
number and references
NFW-F GACTCCTACGGGAGGC
16S rDNA
522Rv-R GCGGCTGCTGGGAC
GP-PB CTGAYSSAGCAACGCCGCG Gram-positive (Bispo et al.)
GN-PB CCTGAYSCAGCMATGCCGCG Gram-negative
HFE_ F GATGACCAGCTGTTCGTGTTC HFE NM 139004.2
HFE_R CCACATCTGGCTTGAAATTCT This study
blaSHV-F ATGCGTTATACGCCTGTG blaSHV (Monteiro et al.)
blaSHV-R TGCTTTGTATTCGGGCCAA Cephalosporin
blaTEM-F TGCCGCATACACTATTCTCAGAATGA blaTEM (Monteiro et al.)
blaTEM-R ACGCTCACCGGCTCCAGATTTAT Cephalosporin
blaCTX-F ATGTGCAGYACCAGTAARGTKATGGC blaCTX (Monteiro et al.)
blaCTX-R TGGGTRAARTARGTSACCAGAAYCAGCGG Cephalosporin
blaKPC-F ATGTCACTGTATCGCCGTCTAGTTC blaKPC EU 784136
blaKPC-R CAATCCCTCGAGCGCGAGTC Carbapenem (Monteiro et al.)
blaIMP-F GAATAG(A\G) (A\G)TGGCTTAA(C\T)TCTC blaIMP (Mendes et al.)
blaIMP-R CAAAC(C\T)ACTA(G\C)GTTATC Carbapenem
blaVIM-F GTTTGGTCGCATATCGCAAC blaVIM (Mendes et al.)
blaVIM-R AATGCGCAGCACCAGGATAG Carbapenem
blaSPM-F CTAAATCGAGAGCCCTGCTTG blaSPM AJ 492820
blaSPM-R CCTTTTCCGCGACCTTGATC Carbapenem (Mendes et al.)
mecA-F AAAACTAGGTGTTGGTGAAGATATACC mecA (Zhang et al.)
mecA-R GAAAGGATCTGTACTGGGTTAATCAG Methilicin
vanA-F GGGAAAACGACAATTGC vanA (Dutka-Malen et al.)
vanA-R GTACAATGCGGCCGTTA Glycopeptide
vanB-F ATGGGAAGCCGATAGTC vanB (Dutka-Malen et al.)
vanB-R GATTTCGTTCCTCGAC Glycopeptide
Acknowledgments
Research supported by FAPESP and CNPq.