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Técnicas microbiológicas rápidas para dirigir el antibiótico empírico precozmente

Patricia Muñoz, MD. Ph.D.

Microbiología Clínica-Enf. Infecciosas

Hospital General Universitario Gregorio Marañón Universidad Complutense of Madrid. Spain

Jueves. NOV 10, 2011 19:15 - 19:35 h

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Presentation and Disclaimer

Presentation Clinical Microbiology and Infectious Diseases

how molecular methods are used today in sepsis management

Disclaimer Advisory boards and Conference honoraria: Pfizer, MSD,

Novartis, Astellas, Biomerieux Research funds: Novartis, Astellas

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Molecular methods (MS) in the Diagnosis of Sepsis

I. Sepsis challenges

II. Role of MM in blood samples

III. Role of MM in positive blood cultures

IV. Role of MM in other samples

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I. Is diagnosis of sepsis still challenging?

Evolution of significant BSI episodes/1000 admissions H. General Universitario Gregorio Marañón

1615,3

16,417,3

21

23,1

21,422,8 23,3

21,8

26,427,9

25,7 25,9 25,726,8 26,7

29,4 29,4

26,8

28,5

31,2

32,8 3334,1

29,8

0

5

10

15

20

25

30

35

40

85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 06 07 08 09 10

Gentileza de Dra. Marta Rodríguez-Creixéms

Total number BSI episodes: 35,161

28,414 patients

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7,76,8

13 12,8

11,6

9,7 9,58,5 8,5

12,511,5

10,9

12,611,711,6

13

10,5

12,711,8

8,59,5 9,3

8

10,4

12,4

0

15

85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 06 07 08 09 10

Gentileza de Dra. Rodriguez Créixems

% polymicrobial BSI episodes

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Microorganism Nº BC Median IQR*

Gram (-) 1,089 8.8 h 4.3 – 15.5

Gram (+) 1,114 11.02 h 6.2 – 17.2

Anaerobes 67 27.8 h 17.7-56.4

Candida sp 88 33.3 h 19.9-47.4

Time to growth of different microorganisms

Gentileza de Dra. Marta Rodríguez-Creixéms

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CHALLENGE FOR MM

We know that

>30% empiric Abs are incorrect

Mortality 7.6% > /hr delay after hypotension

We do not want to answer the question

Did the physician choose the correct empiric therapy?

We want to answer the question

Which antimicrobial agent is best for treating this patient ‘s infection now?

Tenover FC. Ann NY Acad Sci 2010

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Challenges for MM

1. Is this sepsis?

2. Is it caused by a resistant pathogen?

3. Which pathogen?

4. Are there any complications?

Dickermann Am Heart J 2007; Bouza E CID 2004:39;

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IS THIS SEPSIS? Can MM help in the empirical phase of sepsis management?

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Sepsis remains a clinical diagnosis

Multi-symptom manifestation

Common signs: unspecific

Fever, Elevated ESR or CRP

Specific signs: LATE

Hypotension

Lactate> 4 mmol/L

BIOMARKERS AND BLOOD CULTURES

>50% of sepsis have negative BC

NO rapid, S and Sp laboratory tests

Blood cultures usually the base of Dx

PROS

Available in most laboratories

Efficacious and well-known technology

Etiology and susceptibility

Strains storage

Cheap

CONS

30-50% of negative BC

Diagnosis delay may increase mortality

Blood cultures

<50% of the etiological diagnosis of sepsis

+3-4d

Identification and susceptibility

+ 2d

Culture and preliminary data

Bouza, Sousa et al. (2004). CID 39:1170-3

~7h-5d

BC + Gram stain

G- 8.8h ; G+ 11 h

Molecular methods

Different microorganisms Resistance genes Fastidious microorganisms

PCR

Typing

Identification

Resistance genes

Rapid identification Resistance genes

Commercial or in-house methods Pathogen specific, Broad range , Multiplex Different post PCR detection methods

Universal PCR + sequencing

Specific PCRs

Dr. Marin

Molecular methods: blood samples

PCR

Septifast® (Roche)

SepsiTest® (Molzym)

VYOO® (Sirslab)

BlacKLight Sepsis® (Blackbio)

Plex-ID® (Abbott)

Low concentration of microbial DNA High concentration of inhibitors Do not detect fastidious m.s. Detect DNAemia (contaminating?)

Limited susceptibility information No storage of strains

Contamination risk Laborious. Trained personnel Special equipment. Expensive

Rapid results Theoretical high sensitivity Multiple + difficult pathogens Diagnosis in treated patients

PROS

CONS

Dr. Marin

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Some clinical experience with Septifast 23 microorganisms

Patients n +BC +PCR Combined Concordance % pts significant

improved Dx

Mancini N1 Neutropenic 34 20.4% 33% 34% 83% 5.8%

Varani S3 Neutropenic 100 29.2% 21.5% 33.8% 79.2% 4.6%

Louie RF7 General 200 18.5% 22.5% 30% 88.5% 25%

Westh H8 General 359 17% 26% 31.5% 79% 16.7%

Maubon D9 Malignancy 110 29% 25.4% 30% 70% 10%

Avolio M10 ER 144 29.9% 27.8% 37% 56.6% NR

Dierkes C11 General 101 21% 27.4% 35% 77% 8%

Lehman LE12 General 436 21.2% 28% 36.4% 58.2% 29%

Wallet F 13 ICU 72 10% 15% 21% 83.3% 5.5%

Lamoth F14 Neutropenic 86 25% 25% 43% 65% 37%

Tsalik EL15 General ER 306 25% 17.3% 34.2% 82.6% NR

Lilienfeld16 Neutropenic 70 28.5% 21% 38.6% 71% NR

1.JCM 09; 2 Steinmann J. TID 09; 3 J Infect 09; 4. Mussap M. J Chemother 07; 5. Paolucci M. J Med Microb 09; 6. Vince A. J Med Microb 08; 7. CCM 08. 8. JCM 09; 9. J Infect 10. 11. BMC Infect Dis 09; 12. CCM 09; 13. CMI 09. 14. JCM 10; 15. JCM 10; 16. von Lilienfeld-Toal M. JCM 2009

467 episodes sepsis BC +: 21.2%

PCR + 28%

35% empirical therapy: incorrect

PCR+: reduction of inadequate treatment days 22.8 d/100 tests

36.4 d/100 tests in ICU and surgical wards

Tx change: 29% pts

High rate of FN PCRs (64 episodes)

17 Lehmann LE. Crit Care Med 2009; 37:3085–3090

46/135 Ab

changes more rapid

142 Severe Sepsis

BC +: 16.5%

PCR + 34.7%

Only PCR+ vs PCR-

Higher disease severity

Higher + biomarkers

Higher mortality

PCR- no withdrawal of ABS

18 Blood F. Intensive Care Med 2010

BC+: 70% PCR+

PCR+: 21% BC+

SepsiTest (Molzym Co.)

Universal 16S/18S rRNA Gene-Based PCR and sequence analysis

Time-consuming

Bacteria and yeasts

217 Euros

Wellinghausen N. J Clin Microbiol 2009; Lehman Med Microbiol Immunol

187 patients

BC +:18% PCR+: 31.5%

S 87% Sp 85.8%. Concordance 86%

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Test Studies in

sepsis

m.os Resistance

data

Hands-on

time

Time

to dX

COST

SeptiFast® (Roche)

23 mecA 4

SepsiTest® (Molzym)

At least 54 B 5 F

(345 m.os)

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VYOO® (Sirslab)

33 B and 6 F mecA, Van A;B,C,

ESBL SHV

6-8

Plex ID Bacteria and

fungi

6-8

BlacKLight Sepsis® (Blackbio)

All bacteria?? 4

Bad or non-available data Intermediate Good data

21 Current Opinion in Infectious Diseases 2010

BC+

18 h

Transport

Working hrs SC

Culture

SepsiTest®

SeptiFast®

VYOO®

Plex-ID®

Susceptibility

ID

D2 D3 D1

False negatives

Inhibition with WBC >30000/mm3

Specificity of primers and probes

Genetic variability of target site

Sample volume

Not in the list

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False negative PCR

Which should be the ‘gold standard’? BC??

MM positive/ BC negative FP?

Clinically relevant? Many, later confirmed in other samples.

Non viable organisms? Cell-free DNA released from remote infection sites? Antibiotic interference?

MM neg/ BC neg. Unculturable pathogens? Not sepsis???

Refence method should include multiple data 23

Ecker DJ. Expert Rev Mol Diagn 2010

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Are molecular methods of any benefit when BC are already

positive?

Molecular methods from + BC

PCR

+

MALDI-TOF MS (Bruker, Shimadzu, Vitek-MS)

PCR/ESI-MS (Plex-ID®)

PCR and microarray hybridization (Prove-it®)

RealT- specific PCR (Genexpert®, GenOhm®)

Peptide Nucleic Acid FISH (PNA FISH®)

PCR- reverse hybridization (Genotype®)

2-6 h

From POS BC

Malditof (Bruker, Saramis- Shimadzu, Vitek-MS)

Matrix-assisted laser desorption ionization time-of-flight

Positive BC (>107 cfu/mL): < 2h (1-2 min from a colony). Other samples

Problems with S. pneumoniae, S. mitis

> 80% results next day after sample entry

26 La Scola. PLoS ONE 2009; Stevenso LG. JCM 2010; Prod’hom JCM 2010; Risch M. Swiss Med Wkly 2010; Greub G. CMI 2010; Moussaoui W. CMI 2010. Drancourt M. CMI 2010; Emonet S. CMI 2010.

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Clinical experience with Malditof

Samples n Correct

identification Comments

La Scola B. 2009 BC - Bactec 9240 562 76 G +/- 67%/94%

Stevenson LG. 2010 BC - Bactec 9240 212 80 Poor S mitis

Prod’hom G. 2010 BC - Bactec Plus 126 78.7 Poor S pn

Ferroni A. JCM 10 BC - Bact/Alert 312 91 S.a. vs CNS 100%

Christner M JCM 10 BC - Bactec Plus 304 95 S.a better AN bottles

Risch M. 2010 BC, urine, genital, wounds,

etc 204 87 Poor S pn

Moussaoui W. 2010 BC- Bactec 9240 503 90 G +/- 89%/91%

Ferreira L. 2011 BC- Bactec 9240 330 NR G +/- 31.8%/83%

Candida

Malditof potential

Different samples Fungus, virus, parasites Genotyping (nucleic acids)

Resistance R marker protein Degradation of Ab Induced R marker

Virulence Quantification?

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RESEARCH IS ONGOING

Malditof

Some problems to solve

Poor detection of mixed populations

22 cases: only 1 species detected in 18

Sometimes lack of identification or false species

Poor detection of S. pneumoniae (4/20)

Agglutination test in all viridans strep identification (Bruker)

29 La Scola B. PLoS ONE 2009

PCR of several genes • Broad range primers • Polymicrobial inf

4-6 hours Different samples Quantification (sputum)

Typing, virulence, resistance

Biowarfare agents, infection control, syndromic panels

Ecker DJ. Expert Rev Mol Diagn 2010; Emonet S. CMI 2010; Kaleta EJ. JCM 2011.

Electrospray ionization mass Spectrometry

(PCR/ESI-MS) Plex ID® (Abbot)

PROVE-IT™ SEPSIS (MobiDiag)

PCR and microarray (gyrB, parE, mecA, 50 bacterial species, Fungi)

3318 BC / 2107 + (14% non detectable by Prove-It)

S 94.7 %; Sp 98.8% (100% MRSA)

1.6% FP, really FP?

18 h faster than BC

Tissari P. Lancet 2010; 375

MM as a complement to Gram staining of flagged BC? Pathogen directed

Reduce time to effective therapy

Enterococcus PNA FISH

Identification E.faecalis: 1.1 vs 4.1d E. faecium 1.1 vs 3.4

30-d mortality: 26% vs 45%

Hospital savings/yr: $ 20,000

S. aureus or Candida PNA FISH

GeneXpert MRSA-SA-BC® (Cepheid)

Specific RT-PCR

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Ecker DJ. Expert Rev Mol Diagn 2010; Forrest GN. AAC 2008; Zhang N. JID 2010; Ly T. Ther Clin Risk Manag 2008: Hansen WLJ. JCM 2010

C. glabrata and/or C. krusei

C. albicans and/or C. parapsilosis

Molecular detection in Positive blood cultures GeneXpert MRSA-SA-BC® (Cepheid)

406 blood cultures with clusters G + cocci Identification of MSSA and MRSA in 1h Simple. No special requirements

Wolk DM. J.Clin.Microb.2009

Sen 98.3% Spec 99.4% PPV 96.6% NPV 99.7% Contaminants CNS increase cost

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Test Clinical

data

m.os Resistance

data

Hands-on

time

Time To

dX

COST

GENEXPERT aureus ® , Staph Plex

S. aureus MS and MR

PNA FISH

GENOTYPE® 15G+, 17G-, mecA, VanA,B

PROVE IT® 60 B mecA

MALDITOF B, Fung, Micob

Bad or non-available data Intermediate Good data

35 Current Opinion in Infectious Diseases 2010

BC+ Transport

Working hrs SC

Culture Susceptibility

ID

MALDI-TOF®

Prove-It®

MolYsis®

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Does MM help in providing diagnosis of septic patients from

other samples?

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MM in heart valve tissue FIRST-CHOICE technique

Broad-range bacterial or fungal PCR + sequencing Always confirm a positive result (PCR in different sample,

specific PCRs)

Organism-specific PCRs Tropheryma whipplei, B. quintana, B. henselae, C. burnetti

MRSA, S. pneumoniae, Generic PCR sodA for Streptococcus and Staphylococcus

Tang. JCM 2009; Raoult JCM 2005; Fournier CID 2010

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S 96%, Sp 95.3%, NPV 98.4%, PPV 88.5%

Median response time

PCR result (pos or neg): 6 h from the reception of the sample

Hands-on time: 1.5 h

Species level: 24-48 h (6 hrs if targeted PCR used)

Medicine 2007;86:195–202

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Embolus and clots

Vegetations that have embolized to peripheral arteries

Septic metastases

Pleural fluid

International Journal of Infectious Diseases (2009)

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Culture 16S PCR p

Sensitivity 56.1% 81.7% <0.001

Specificity 97.4% 99.1% 0.057

Positive predictive value 76.7% 94.4% 0.004

Negative predictive value 92.8% 96.8% 0.008

Gram stain

Sensitivity

Specificity

24.4%

99.5%

723 pleural fluids

S. Insa, E. Bouza. Medicine (Baltimore) 2011.

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Challenges for MM

1. Is this sepsis?

2. Is it caused by a resistant pathogen?

3. Which pathogen?

4. Are there any complications?

Dickermann Am Heart J 2007; Bouza E CID 2004:39;

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Etiologic Dx of endocarditis LightCycler SeptiFast ®

63 IE

41 + BC: 11 positive SF (26.8%)

5/9 S. aureus, 0/9 CNS

1/7 Enterococcus

5/12 Streptococcus

0/4 other microorganisms (2 E. coli, 1 P. mirabilis, 1 C.

parapsilosis)

3 + in 22 BC – endocarditis S. gallolyticus, S. aureus, and E. faecalis

J. P. Casalta. Eur J Clin Microbiol Infect Dis (2009)

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IE: specific etiology

Gram neg

40%Gram pos

53%

Anaerobes

3%

Fungi

4%

HOSPITAL GENERAL UNIVERSITARIO GREGORIO MARAÑÓN.

1,826 EPISODES OF BSI IN 2009

Fungi

1.9%

Gram neg

5.5%

CNS

17%

Enterococcus

12.1%

Streptococcus

spp

23.8%

S. aureus

26.5%

Unknown

9.9%

ENDOCARDITIS

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Let’s imagine ….

1000 pts with SS

400 BC+ MM in blood 75% S 1-3 d more rapid ID

600 BC-

Septifast 30%S

20% Qx or other fluids

Specific PCR in blood <1%

>800 patients would benefit from MM methods For 300 the impact would be very major

180 pts

>300 pts?

120 pts

6 pts

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Resistance in clinical samples Too many different techniques. Which is the best? Indications for specific populations. Complement to

BC and in selected high risk patients ? More microorganisms should be detected, more

information on resistance provided Quantification?

Evaluation in different clinical settings and cost-efficiency studies are warranted

Wishes for the future

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Challenges for MM

1. Is this sepsis? Partially yes, if positive

2. Is it caused by a resistant pathogen? Not enough yet

3. Which pathogen? Partially yes

4. Are there any complications? Not enough yet

May not yet be the definite solution, but I think they are already helping…!!!

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Will New Diagnostic Solutions Help Meet the Challenges of Sepsis?

I think so!