Diagnosis of Infection in Sepsis

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Diagnosis of infection in sepsis: An evidence-based review

Jonathan Cohen, MB, FRCP; Christian Brun-Buisson, MD; Antoni Torres, MD; James Jorgensen, MD

I nfection is a sine qua non of sep-sis. Sepsis can complicate infec-tion occurring at any site, mostcommonly the respiratory tract,

abdomen, and bloodstream. Greater than90% of cases of sepsis are caused by bac-teria, and Gram-negative and Gram-positive organisms occur with approxi-mately equal frequency, although Gram-positive organisms are becoming morecommon. Fungi, in particular Candidaspecies, are sometimes responsible, but a wide variety of other organisms have oc-casionally been implicated (1, 2).

There are several reasons why it isimportant to try and make a microbiolog-ical diagnosis in septic patients. First, andmost important, is to ensure that effec-tive antimicrobial therapy is given. Thereis good evidence to support the intuitive

belief that patients given appropriatetherapy are more likely to survive thanthose given inadequate or inappropriatetreatment (reviewed in Reference 3). Sec-ond, obtaining microbiological informa-tion will ensure that the local epidemio-

logic database is current, without whichlogical prescribing is difcult, if not im-possible. There are substantial differencesbetween intensive care units (ICUs) in themicrobial ecology, including, for exam-ple, the prevalence of methicillin-resis-tant Staphylococcus aureus and vanco-mycin-resistant Enterococcus faecium or Enterococcus faecalis . Antimicrobial re-sistance patterns, too, vary widely: exam-ples include penicillin and multiple drugresistance in Streptococcus pneumoniaeand third-generation cephalosporin or

uoroquinolone resistance in Enterobac-teriaceae. Furthermore, these patternsare constantly changing, and an up-to-date awareness of these patterns is obvi-ously essential when considering empiri-cal therapy. Finally, knowledge of themicrobial etiology of sepsis may be im-portant in the choice of adjunctive ther-apies. This is not yet a clinical reality but will clearly be important if, for instance,antiendotoxin agents ever enter the clin-ical arena.

We will rst consider the general ap-proach to the diagnosis of infection inseptic patients and then consider someaspects of infections at major sites. Ourintent is to focus on the microbiologicalaspects of diagnosis, although where ap-propriate, we also comment on other mo-dalities, such as imaging. The clinicalmanagement of these infections is dis-cussed elsewhere.

Methods

A computer-based review of the liter-ature was undertaken using Medline from1991 until September 2003 as the pri-mary database. The references obtained were searched manually for relevance.Other recent clinical articles were identi-ed by manual search of the relevant

journals . Although the li teraturesearches were not extended backward be-fore 1991, key earlier articles are fre-quently cited. In reviewing this eld, wefrequently observed that whereas there isa large literature on the microbiologicalaspects of infection in general, there is apaucity of data concerning sepsis in par-ticular. Hence, we have often beenobliged to make recommendations basedon the available literature and our ownclinical experience and that of others.

From the Department of Medicine, Brighton &Sussex Medical School, Brighton, UK (JC); the MedicalIntensive Care Unit, Hopital Henri Mondor & UniversitéParis XII, Creteil, France (CB-B); the Institut Clínic dePneumologia i Cirurgia Toràcica, Hospital Clínic deBarcelona, Spain (AT); the Department of Pathology,University of Texas Health Science Center, San Anto-nio, TX (JJ).

Copyright © 2004 by the Society of Critical CareMedicine and Lippincott Williams & Wilkins

DOI: 10.1097/01.CCM.0000145917.89975.F5

Objective: In 2003, critical care and infectious disease expertsrepresenting 11 international organizations developed manage-ment guidelines for the diagnosis of infection in sepsis that wouldbe of practical use for the bedside clinician, under the auspices ofthe Surviving Sepsis Campaign, an international effort to increaseawareness and improve outcome in severe sepsis.

Design: The process included a modied Delphi method, a con-sensus conference, several subsequent smaller meetings of sub-groups and key individuals, teleconferences, and electronic-baseddiscussion among subgroups and among the entire committee.

Methods: The modied Delphi methodology used for gradingrecommendations built on a 2001 publication sponsored by theInternational Sepsis Forum. We undertook a systematic review of theliterature graded along ve levels to create recommendation gradesfrom A to E, with A being the highest grade. Pediatric considerationsto contrast adult and pediatric management are in the article byParker et al. on p. S591.

Conclusions: Obtaining a precise bacteriological diagnosisbefore starting antibiotic therapy is, when possible, of para-mount importance for the success of therapeutic strategyduring sepsis. Two to three blood cultures should be per-formed, preferably from a peripheral vein, without intervalbetween samples to avoid delaying therapy. A quantitativeapproach is preferred in most cases when possible, in partic-ular for catheter-related infections and ventilator-associatedpneumonia. Diagnosing community-acquired pneumonia iscomplex, and a diagnostic algorithm is proposed. Appropriatesamples are indicated during soft tissue and intraabdominalinfections, but cultures obtained through the drains are dis-couraged. (Crit Care Med 2004; 32[Suppl.]:S466–S494)

K EY WORDS: sepsis; infection; evidence-based; diagnosis; mi-crobiological; laboratory

S466 Crit Care Med 2004 Vol. 32, No. 11 (Suppl.)



The search strategies used were as fol-lows.

● Bacteremia : The primary search termsused were bacteremia/septicemia (diag-nosis, microbiology) and blood (micro-biology) and were combined with sec-ondary search terms, which weresepsis/sepsis syndrome (diagnosis, mi-crobiology), blood specimen collection(methods), bacteriologic techniquesand diagnostic tests, routine (meth-ods). Studies addressing exclusively pe-diatric populations were excluded.

● Central Venous Catheter Infection :The primary search terms used werecatheterization, central venous (adverse effects), and catheters, indwelling(adverse effects) and were combined with secondary search terms, which were sepsis/sepsis syndrome (diagno-sis, microbiology, etiology), bactere-mia/septicemia (diagnosis, microbiol-ogy, e tiology) , blood specimen

collection (methods), bacteriologictechniques and diagnostic tests, rou-tine (methods).

● Ventilator-Associated Pneumonia : Theprimary search terms used were respi-ration, articial (adverse effects) venti-lation, mechanical (adverse effects) andintensive care (methods) and werecombined with secondary searchterms, which were pneumonia (diagno-sis, microbiology, etiology), sepsis/ sepsis syndrome (diagnosis, microbiol-ogy, etiology), bacteremia/septicemia

(diagnosis, microbiology, etiology),bacteriologic techniques and diagnos-tic tests, routine (methods).

● Surgical Site Infections and Intraab-dominal Sepsis : The primary searchterms used were wound infection (di-agnosis, microbiology), abscess (diag-nosis, microbiology), and abdomen and were combined with secondary searchterms, which were sepsis/sepsis syn-drome (diagnosis, microbiology, etiol-ogy), bacteremia/septicemia (diagnosis,microbiology, etiology), bacteriologictechniques, diagnostic tests, routine(methods) and interventional radiol-ogy.

General Considerations in theDiagnosis of Sepsis

There are three key difculties associ-ated with the diagnosis of infection inpatients who have sepsis.

● Establishing Infection as the PrimaryCause : Establishing that the patient

has an ongoing infection and, there-fore, has sepsis rather than a noninfec-tive cause of the systemic inamma-tory response syndrome can beextremely difcult. An important rststep is a systematic consideration of possible noninfective causes. Knowl-edge of other pathologies that maymimic sepsis and how they apply to thespecic patient can make up for a rel-

ative paucity of clinical information inpatients who may be sedated or criti-cally ill (Table 1).

● Localizing the Site of Infection : Thismay be straightforward but frequentlyis confounded by the fact that there aremultiple pathologic processes occur-ring concurrently (especially in ICU-acquired sepsis) or by the frequent useof antibiotics, which undermine micro-biological diagnosis. Occasionally, thesite of infection is occult, typically inpatients with primary bacteremia (non-catheter-related), or when there is si-nusitis or deep intraabdominal infec-tion.

● Interpreting the Microbiological Find-ings : Conventional microbiology hasseveral limitations in hospitalized pa-tients who may be septic (Table 2).Principal among these is the fact thatmany organisms isolated from nonster-ile sites may represent either coloniza-tion or infection; microbiology alonecannot answer this question. Con- versely, the microbiology laboratorymay report negative ndings in sam-ples from sites that are, in fact, in-fected, either because antibiotics havesterilized the specimen or because spe-cial procedures need to be done (e.g.,immunouorescence to detect Pneu- mocystis carinii ).

A Clinical Approach. Fever is a com-

mon sign in hospitalized patients and willoften be the rst indication of sepsis.Practice guidelines for the evaluation of fever in the ICU have been published (4).Focused clinical examination, guided byrisk factors relevant to the individual pa-tient, will often reveal potential sourcesof sepsis and guide subsequent investiga-tions. Surgical and traumatic woundsshould be exposed and examined for signs

of infection. Particular attention shouldbe paid to vascular access sites for signsof phlebitis or cellulitis and to pressureareas or injection sites for evidence of softtissue infection. Evidence of sinusitisshould be sought, and funduscopy is in- valuable in detecting candidal endoph-thalmitis, a pathognomonic feature of systemic fungal sepsis. Urine in the cath-eter may be frankly purulent, and thepresence of diarrhea may indicate Clos-tridium difcile -associated colitis. Theimportance of repeated, complete physi-

cal examination to detect the emergenceof new signs cannot be overstated. Nonspecic Markers of Infection. Tra-

ditional markers of infection, such asneutrophilia, lack sufcient sensitivityamong hospitalized patients to be of value in distinguishing sepsis from non-infective inammatory processes. Forthis reason, investigators have longsought alternative markers that could actas surrogate evidence of positive micro-biology, which for a number of reasonsmay not be readily available. These sur-

rogate markers need to have several char-acteristics: the main requirements arethat they should be cheap, easy, andquick to assay and that they should havea high sensitivity and specicity. Amongthe most commonly studied of these po-tential markers are C-reactive protein,procalcitonin, interleukin-6, and inter-

Table 1. Noninfective causes of systemic inammatory response syndrome

Tissue injury Surgery/traumaHematoma/venous thrombosisMyocardial/pulmonary infarctionTransplant rejectionPancreatitisErythroderma

Metabolic Thyroid storm Acute adrenal insufciency

Therapy related Blood productsCytokines, especially granulocyte-macrophage colony-stimulating factor Anesthetic-related malignant hyperpyrexia, especially halothaneNeuroleptic malignant syndrome, e.g., caused by haloperidolOpiates/benzodiazepines

Malignancy Hypernephroma/lymphomaTumor lysis syndrome

Neurologic Subarachnoid hemorrhage

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leukin-8, as well as a range of more com-plex immunologic and endothelial mole-cules (5, 6) .

Levels of procalcitonin and C-reactiveprotein are straightforward to assay andcorrelate well with the degree of inam-matory response. Procalcitonin may havesome advantages over C-reactive proteinin that it rises more quickly at the onsetof inammation and is cleared morequickly as inammation resolves (7). Lev-els of procalcitonin correlate moreclosely with the severity of sepsis (8). In aprospective study of ICU patients, de Werra et al. (9) found that procalcitoninlevels of 1.5 ng/mL or more had a sensi-tivity of 100% but a specicity of 72% inidentifying sepsis. Such markers, there-fore, cannot alone differentiate sepsisfrom other causes of systemic inamma-tory response syndrome; rather, they area part of a systematic evaluation that in-cludes clinical examination and directeddiagnostic techniques. Daily, sequentialmeasurement of inammatory markers islikely to be of more value for the diagno-sis of infection than one-off measure-ments (6).

Detection of circulating endotoxinmight be expected to be a specic test forsepsis resulting from Gram-negative in-fection. For many years, the only testavailable has been the Limulus amebo-cyte lysate assay. Most (10) but not all(11) investigators have found that endo-toxemia does correlate with Gram-negative infection and outcome, but de-spite many attempts, the assay has neverbeen found to be robust and reliableenough for routine clinical use (12). Veryrecently, a new assay has been developed

based on the detection of endotoxin-induced neutrophil chemiluminescence(13). Preliminary data suggest that the negative predictive value of this test issufciently high that it might be used torule out Gram-negative infection (13a).

Bloodstream Infection

Question: Are blood cultures required in all patients suspected of severe sepsis?

Yes; Grade E

Recommendation: All patients with sus-pected severe sepsis should have bloodcultures taken, whatever the suspectedsource, although in some cases (such ascellulitis), the yield is low. Althoughstrict evidence-based medicine criteriasuggest that this recommendation wouldonly merit a grade E, it is evident that theneed to take blood cultures in suspectedsepsis would universally be regarded as astandard of care. Fever, chills, hypother-mia, leukocytosis, left shift of neutro-phils, neutropenia, and the developmentof otherwise unexplained organ dysfunc-tion, e.g., renal failure or signs of hemo-dynamic compromise, are specic indica-tions for obtaining blood for culture.Blood cultures should be taken as soon aspossible after the onset of fever or chills.In patients with suspected catheter-related infection, a pair of blood culturesobtained through the catheter hub and aperipheral site should be obtained simul-taneously. When severe sepsis develops inpatients colonized by Candida species attwo or more sites, blood cultures shouldbe taken for the detection of candidemia;it is uncertain if the lysis-centrifugation

method increases the sensitivity for de-tecting candidemia in this setting.

Yes; Grade E

Rationale : The incidence of sepsis andbacteremia in critically ill patients hasbeen increasing in the past two decades(14, 15). Thirty percent to 50% of pa-tients presenting with a clinical syn-drome of severe sepsis or shock have pos-itive blood cultures. Therefore, bloodshould be obtained for culture in anycritically ill septic patient. While it re-

mains difcult to predict bacteremia inpatients with sepsis (16), a number of clinical and laboratory parameters are in-dependently correlated with the presenceof bacteria in the blood of patients wheninfection is suspected. These includechills, hypoalbuminemia, the develop-ment of renal failure, and a diagnosis of urinary tract infection (17, 18); other cri-teria are new fever, hypothermia, leuko-cytosis and left shift of neutrophils, neu-tropenia, and signs of hemodynamic

compromise (19). Peaking fever appearsto be more sensitive than leukocytosis topredict bacteremia (20); however, feverand low-grade bacteremia can be contin-uous, such as in endocarditis.

The proportion of bacteremic septicpatients varies markedly according to thesource of infection. The major sources of bacteremia within the context of severesepsis or shock are pneumonia and intra-

abdominal infection (21, 22); othersources generally account for 5% of cases. Because pneumonia is a majorsource of severe sepsis, lower respiratorytract infections account for a substantialproportion of all bacteremias recorded insuch patients. However, blood culturesare neither sensitive nor specic in thediagnosis of pneumonia. Between 3% and12% of bacteremias occurring in ICU pa-tients will have a respiratory tract source(23–25), but fewer than one quarter of cases of ventilator-associated pneumonia

(VAP) are associated with bacteremia(26). It is not uncommon that patients with ICU-acquired sepsis have severalsources of infection, and bacteremia inpatients with suspected VAP, in fact, oftenarises from outside the chest (26, 27).Meduri et al. (27) demonstrated that twothirds of patients with nosocomial pneu-monia will have at least one other focusof infection, usually urinary or central venous catheter (CVC) related. For thisreason, published expert opinion is thatblood cultures are an essential part of the

workup of a patient with suspected VAP,although not in themselves diagnostic of VAP (28).

Supercial skin and soft tissue infec-tion rarely cause sepsis and uncommonlycause bacteremia. Although making up to

5% of all causes of bacteremia amonghospitalized patients (14, 24), deep super-cial skin and soft tissue infections andlocalized intraabdominal sepsis are fre-quently associated with bacteremia. Fur-thermore, empirical antibiotics may needto be given before samples from the sus-

pected site of infection itself are available.However, such deep infections are fre-quently polymicrobial, comprising fecalorganisms, and blood cultures may notidentify the full range of organisms involved, particularly anaerobes.

Question: Does the technique used in ob-taining blood cultures inuence the sensitivity and specicity of this investiga-tion?

Yes; Grade B

Table 2. Problems with interpretation of microbiology in hospitalized patients

Distinguishing colonization from infectionPrior antibiotic therapyCorrectly identifying unfamiliar organismsDetermining the signicance of polymicrobial culture resultsInterpreting the importance of organisms normally of low virulence




Recommendation : Blood should be ob-tained for culture from a peripheral vein whenever possible, and a protocol forskin preparation and sampling should beadopted to minimize the risk of contam-ination. The blood culture stopper shouldalso be disinfected before inoculation. In-oculating at least 10 mL of blood perbottle (at least 20 mL total per culture) isrecommended to optimize the sensitivity

of cultures. If insufcient blood is avail-able or if anaerobic infection is unlikelygiven the clinical context, only the aero-bic bottle should be inoculated.

Grade B

Rationale : When a decision has beenmade to take blood for culture, adherenceto a protocol for obtaining the specimenresults in lower contamination rates andimproved yield (19, 29). The cost of addi-tional investigations, treatment, and in-patient stay associated with each contam-inated blood culture has been estimatedto be between $1,000 and $5,000 (30, 31).Furthermore, with Gram-positive organ-isms making up an increasing proportionof signicant blood culture isolates, iden-tifying such isolates as contaminants ismore difcult than ever (32).

Blood should be obtained by fresh ve-nepuncture whenever possible. Sites as-sociated with skin contamination (e.g.,femoral site) or loss of skin integrity (e.g.,burns or dermatological disease) shouldbe avoided. Denitive evidence that theskin disinfection technique reduces bloodculture contamination rates is lackingbut can be inferred from the ndings of controlled trials that demonstrate supe-riority of one skin preparation agent overanother. The relevant trials since 1990are summarized in Table 3 (33–35). Onetrial compared a chlorhexidine-alcohol

solution with povidone-iodine and foundthe former associated with a 60% lowercontamination rate (34). A second studyfound unexpectedly low contaminationrates in both patient groups. Three othertrials showed no benet of one agent overthe other. One study concluded that con-tamination occurs during laboratoryspecimen handling (33). In one recenttrial comparing four antiseptics in a

crossover trial during 12-wk periods, theoverall contamination rate was 2.6% and varied from 2.9% to 2.5% across differentprotocols; alcohol-based antiseptics wereslightly but not signicantly better (35).Overall, alcohol-based preparations ap-pear to provide lower contaminationrates, but it is unclear that these aresuperior to alcohol alone (33, 35). Theconventional view is that skin should beswabbed twice with an antiseptic solu-tion; however, recently available alcohol-based antiseptics may be more efcientthan nonalcohol-based solutions, and oneswabbing may sufce (34).

The blood culture stopper should alsobe disinfected before inoculation of theblood sample. While we are aware of nodata that directly address this issue, it isthe recommendation of previously pub-lished expert opinion (36). Contrary toearlier reports (37, 38), a recent meta-analysis suggested that contaminationrates are lower if a needle change is per-formed (31). However, given the associ-ated risk of needle-stick injury to person-nel, this should be avoided.

Inoculation of three colony-formingunits (cfu) into blood culture is requiredto give 100% culture positivity (39). Theconcentration of bacteremia in adult pa-tients is frequently below one viable or-ganism per milliliter (40). For these rea-sons, it is not surprising that the volume

of blood inoculated into culture is animportant variable determining culture yield (19, 41). This effect has also beendemonstrated in clinical studies, al-though not specically in sepsis patients(42, 43). While the blood culture systemused will determine the volume of bloodthat may be used, in adults, a minimumof 20 mL is required per venepuncture(10 mL per bottle) (36, 44), while increas-

ing the sample volume above 30 mL isnot associated with signicantly im-proved culture rates (19). In infants, in whom bacteremia is associated withhigher cfu/mL, a smaller volume of blood(0.5–2 mL or 1% of circulating blood volume) can be used for culture (45). Although expert opinions diverge on theuse of anaerobic blood cultures (46), an-aerobic organisms only make up about5% of blood culture isolates (47, 48). Fur-thermore, aerobic culture bottles aremore successful in culture of the over- whelming majority of organisms identi-ed in blood (49). Therefore, if insuf-cient blood is available to inoculate twoculture bottles, only the aerobic bottleshould be inoculated.

Ideally, blood cultures should bedrawn before initiating antibiotics, andpatients should not be receiving paren-teral antibiotics when blood cultures areperformed. Otherwise the indications forperforming blood culture are the same,irrespective of whether the patient is re-ceiving antibiotics. In patients receivingantibiotics, expert opinion suggests thatblood cultures should be taken, wherepossible, immediately before a regulardose of antibiotic so that blood levels areminimized (32). In this group of patients,the use of media containing antibiotic-adsorbing substances, such as BacT/AlertFAN or BACTEC Plus/F, should be con-

Table 3. Trials comparing skin disinfection techniques for blood sampling

Protocol Conclusion Reference

10% PVI and 0.2% chlorine peroxide No benet 57 Alcohol then PVI compared with alcohol alone No benet 3370% iodophore compared with tincture of iodine Tincture of iodine associated with 50% fewer contaminated cultures 6070% isopropyl alcohol/70% PVI compared with isopropyl

alcohol, 10% acetone and PVI dispenserIsopropyl alcohol and 10% acetone and PVI dispenser associated

with 50% fewer contaminated cultures61

In ICU patients, 10% PVI vs. 0.5% chlorhexidine 70%isopropyl alcohol (both applied once)

Chlorhexidine-alcohol associated with 60% reduction incontamination rate (1.4% vs. 3.3%; p .004)

34

2% chlorhexidine 70% isopropyl alcohol vs. 2%tincture of iodine 70% isopropyl alcohol

No difference (contamination rate, 1/215 vs. 3/215) 116

70% isopropyl-alcohol vs. 10% PVI, or tincture of iodine,or PVI 70% ethyl-alcohol (all applied three times)

No difference (70% alcohol: 2.50%, PVI: 2.93%, tincture iodine:2.58%, PVI 70% alcohol: 2.46%)

35

PVI, povidone-iodine; ICU, intensive care unit.




sidered, because they are associated withincreased recovery of signicant patho-gens (50 –52). In suspected fungemia,therapy with antibacterial agents clearlyshould not impact on yield.

Question: Is it appropriate to obtainblood for culture through a central catheter or an arterial line?

Yes; Grade D

Recommendation : Blood drawn throughcatheters is at a higher risk of contami-nation than when drawn from a periph-eral vein, and the latter is recommended.Only when peripheral blood is too dif-cult to obtain should only catheter bloodbe cultured, unless catheter-related in-fection is suspected; in the latter case,both peripheral blood and catheter bloodshould be cultured and their time to pos-itivity compared.

Grade D

Rationale : Blood taken from a central ve-nous or arterial catheter is about twicemore likely to be contaminated than pe-ripheral blood adequately sampled (30,53–55), as is blood taken from previouslyplaced peripheral cannulas. However,contamination rates may not be higherfor blood obtained through a peripheralcannula at the time of insertion, and thisis acceptable as a means of minimizingthe number of venipunctures (56). In onerecent study comparing 499 blood cul-tures taken via central venous or arterialcatheters with blood obtained via periph-eral venepuncture (55), of which 426 were negative at both sites, the overallsensitivity was only 78% (positive predic-tive value, 63%) was higher for arterialblood samples (92% vs. 61%), and speci-city was 95% (negative predictive value,98%). The suboptimal specicity may,however, have been confounded by cath-eter colonization/infection. It may bepossible to minimize contamination of blood obtained through venous cathetersby adherence to a meticulous sterile tech-nique (57), but this approach should onlybe used when no site for venepuncture isavailable.

Question: Is there evidence to determine how many sets of blood cultures should be taken at initial evaluation?

Yes; Grade D

Recommendation : Two (maximum three)sets of blood cultures are sufcient andshould be obtained at the initial evalua-

tion of each episode of suspected bacte-remia.

Grade D

Rationale : In practice, as a noninvasive,safe, and low-cost investigation, bloodculture will often be performed whenthere are few specic indications, and toomany blood cultures are actually taken inICU patients (32, 58). When bacteremia is

associated with endocarditis, if one cul-ture is positive, the probability of anysubsequent culture being positive ex-ceeds 95%. In bacteremia associated withother sources of infection, sensitivity ex-ceeding 99% is reached with either twoor three cultures (59). Conversely, thetaking of only one culture is rarely per-missible because the rate of contamina-tion of an individual set of blood culturesis nite, ranging from 1% to 4.5% (33,60, 61). In this setting, interpretation of asingle isolate of a potentially contaminat-

ing organism, especially coagulase-negative staphylococci, may, therefore,be exceedingly difcult (32). Conversely,a single negative blood culture has a 95%probability of excluding bacteremiacaused by nonfastidious species.

Question: Is there evidence that temporal separation of blood cultures is valuable?

No; Grade D

Recommendation : In critically ill septicpatients, in whom it may not be possibleto delay treatment, no interval is required

between taking two or three sets of bloodcultures.

Grade D

Rationale : In patients who are not criti-cally ill, published expert opinion hasbeen that a 30- to 60-min interval shouldbe allowed to elapse between obtainingsets of blood cultures (56). However, theonly published study to directly addressthe efcacy of serial vs. simultaneousblood cultures demonstrated that draw-ing blood cultures simultaneously or at

intervals over a 24-hr period did not af-fect yield (43).The literature contains no clinical

data relating to timing of blood cultures with respect to timing of fever or chills.Nevertheless, bacteria are rapidly clearedfrom blood, and development of feverusually follows an episode of bacteremiaby 30–90 mins. Published expert opinionis, therefore, that blood cultures shouldbe taken as soon as possible following theonset of fever (36).

Question: Are there indications for re- peating blood cultures in a given episode of infection?

Yes; Grade E

Recommendation : Blood cultures can berepeated for the evaluation of a septicepisode when initial cultures are negativeand special culture media not initiallyrequired appear necessary (e.g., sus-

pected tuberculosis, fungal infection). Inaddition, blood cultures may be repeatedin patients having initially positive cul-tures who fail to respond after 48–72 hrsof appropriate antibiotic therapy to doc-ument a complicated course, particularlyinadequate control of the source. In othercircumstances, it is not recommended toperform repeated blood cultures after ini-tiation of therapy.

Grade E

Rationale : Repeated blood cultures using

specic media may be useful if the initialset is negative and there is a suspicion of infection caused by slow-growing or fas-tidious organisms, e.g., tuberculosis orfungal infection. In such instances, cul-tures on specic, more sensitive media ora lysis-centrifugation technique may beused preferentially (62).

In patients having documented bacte-remia, there are two indications for ob-taining repeated blood culture in a givenepisode of infection. One is to assess theeffectiveness of therapy within the rstfew days. This is specically indicated incases of endocarditis and septic thrombo-phlebitis, when continuous blood seedingoccurs, often persisting for a few daysafter the initiation of therapy, and theoptimal serum bactericidal effect isneeded. Likewise, it should be recalledthat the clinical evolution and lack of relapse of a given episode of a bacteremicinfection is the single denitive test of cure (63) and repeated blood culturesmay be obtained in endocarditis after theend of therapy. In other circumstances, itis generally useless to obtain repeatedblood cultures, particularly in nonintra- vascular community-acquired infections.For example, one study found that of 139patients in whom repeated blood cultures were obtained in the rst 3 days of ad-mission and initiation of therapy, onlyone had a positive culture with a differentpathogen from those recovered initially(64).

Question: Is a single positive blood cul-ture of clinical value?




Yes; Grade E

Recommendation : A single positive bloodculture is clinically signicant when notgrowing a usual skin contaminant. How-ever, a single positive blood culture grow-ing one of the common skin contami-nants is unlikely to be of clinical value.

Grade E

Rationale : As mentioned above, the inter-pretation of a single positive blood cul-ture is highly dependent on the numberof culture sets taken at the time of eval-uation and on the technique used forsampling. A single positive blood cultureamong two or more sets taken is usuallyclinically signicant when growing a typ-ical pathogen such as S. aureus , Kleb- siella , Pseudomonas species or Candida .However, even with such organisms, in-terpretation should be cautious if bloodhas been drawn from patients with diffuseskin lesions, which may become heavily

colonized with these organisms and con-taminate sampling.

A single blood culture growing a usualskin commensal (coagulase-negativestaphylococci, Corynebacterium , Bacil-lus species, and viridans group strepto-cocci), especially when drawn from acatheter, may indicate contaminationduring sampling or subsequent handlingof the sample or colonization of the cath-eter hub or catheter infection and canonly be interpreted in light of clinicalinformation. Infection with those species

is usually considered when at least twoblood cultures grow the same species. Indifcult cases, when the patient’s condi-tion is not severe, it is, therefore, prefer-able to repeat blood cultures to conrmthe presence of true bacteremia. A longtime to positivity (i.e., 48 hrs) makesthe likelihood of true bacteremia low, ex-cept possibly in patients receiving antibi-otics. Polymicrobial bacteremia may alsobe associated with contamination, exceptin the context of intraabdominal infec-tion or necrotizing soft-tissue infection.Catheter-related bacteremia may also bepolymicrobial in a substantial proportionof cases; in one study, this proportion wasfound to be inordinately high, at 43%(65).

Central Venous CatheterInfection

Denition of Terms. Unfortunately, inmuch of the literature relative to the di-agnosis of CVC infection, denitions of

catheter-related bacteremia (CRB) andcatheter-related sepsis are not strictly ad-hered to. Catheter infection may causelocal exit-site infection, bacteremia, andnonbacteremic sepsis, and it is clinicallyimportant to distinguish catheter-related bacteremia from nonbacteremic cathe-ter-related sepsis . Adding to the confu-sion is that in many epidemiologic andsome clinical studies, denite CRB are

often combined with “primary bactere-mia.” Primary bacteremia (which by def-inition is from an unknown source) oc-curring in a patient having an indwellingcatheter is often considered catheter-related when caused by pathogens likelyassociated with catheter infection (e.g.,coagulase-negative staphylococci,Corynebacterium , Bacillus species, andCandida species) in the absence of other potential sources (66); however,primary bacteremia from a truly unknownsource is not uncommon in the ICU, andtheproportionof these ascribed to catheter-infection may be overestimated (67).

Denite CRB is dened by the pres-ence of three criteria (66, 68, 69):

● Positive (semiquantitative or quantita-tive) catheter culture

● Positive peripheral blood culture takenbefore catheter removal

● The same microorganism is identiedin 1 and 2

Probable CRB can be dened as bac-teremia plus either:

● Pus at the catheter-insertion site● Catheter hub culture positive● Positive central blood culture growing

5 times more organisms than periph-eral blood or a differential time to pos-itivity 2 hrs

In each case, the catheter should growthe same microorganism as in peripheralblood and no other source for bacteremiashould be identied.

Because catheters can be contami-nated during removal and give a positive

culture when immersed in culture broth,a positive catheter culture has been de-ned (70, 71) as 15 colonies on semi-quantitative culture of the catheter tip(72) or 103 colonies on quantitativeculture (73–75). The different laboratorytechniques used to culture CVCs are beyond the scope of this article and haverecently been reviewed (76).

Nonbacteremic catheter-related sepsis is dened as a positive catheter cul-ture when this is considered to be the

source of the patient’s sepsis but bactere-mia does not occur (70, 74); this diagno-sis by exclusion requires that no otherapparent source explains the sepsis syn-drome and that sepsis abates within 48hrs of catheter removal.

Local catheter infection is dened as apositive (semiquantitative or quantita-tive) catheter culture in the presence orabsence of local symptoms (erythema,

tenderness) and the absence of a systemicinammatory response.

Question: Are there clinical features suggestive of catheter-related infection?

Yes; Grade D

Recommendation : Signs of inammationat the catheter entry site are neither sen-sitive nor specic to suggest infection;most central venous catheter infectionsoccur in their absence, and their pres-ence should not lead to catheter removal.However, when present, a few signs andsymptoms are highly suggestive of cath-eter infection in a septic patient, includ-ing gross purulence at the catheter inser-tion site, cellulitis extending 4 mmaround the site, or tunnel tract infection.Gross purulence at the CVC site shouldprompt catheter replacement at a newsite, irrespective of culture results.

Grade D

Rationale : Most central venous catheter-related infections occur in the absence of local signs suggestive of infection. Con-

versely, local signs of inammation aroundthe catheter skin entry site, including ery-thema, swelling, and tenderness, are quitecommon beyond 48 hrs, and it is possibleto nd marked erythema with no evidenceof systemic infection (61, 77). Local signsare actually poorly correlated with cathetercolonization (72). For example, Safdar andMaki (78) found that among 1,263 central venous catheters, the index of local inam-mation (including pain, erythema, swell-ing, and purulence) did not differ amongnoncolonized catheters, colonized cathe-

ters (n 333), and those associated withbacteremia (n 35), although the lattertended to have a higher index. In one study,erythema extending 4 mm around theskin entry site was found to be signicantlyassociated with catheter infection (79).Therefore, while overt inammation is sug-gestive of infection (68), local signs cannotbe usually relied on to clinically identifycatheter infection. When frank inamma-tion and purulence around a catheter in-sertion site are present, however, a CVC




infection is very likely (70, 77), and expertopinion supports removal of the catheter(68, 69, 80).

Question: What diagnostic tests can beused to investigate and conrm catheter- related infection?

Recommendation : Cultures of cathetersegment and blood are the only means todenitely prove or disprove catheter-

related infection and bacteremia. Whencatheter infection is suspected, swabsshould be taken from the insertion sitefor culture. Such samples have high neg-ative predictive value for systemic infec-tion, but their positive predictive value islow, especially when coagulase-negativestaphylococci are recovered, unless semi-quantitative cultures are obtained. Rou-tine surveillance cultures of the catheterinsertion site are not recommended. Hubcultures may be performed for long-termcatheters and those used for parenteral

nutrition, as the frequency of intralumi-nal colonization increases with the cath-eter indwelling time. Their predictive value is comparable with that of skin sitecultures. When bacteremia is suspectedand the catheter can be left in place (nofrank local signs, no severe sepsis), bothperipheral and catheter-blood culturesshould be performed and their differen-tial time to positivity measured.

Grade D

Rationale : Most catheter infections in

ICU patients arise from colonization atthe skin insertion site and along the sub-cutaneous tract of the catheter (81).Hence, skin insertion site cultures shouldhave a good predictive value for catheterinfection. Few studies have investigatedskin insertion sites cultures in ICU pa-tients (82). In patients with long-termnutrition catheters, negative site cultureshad a negative predictive value for lineinfection of 98% (83), whereas positiveculture, particularly of organisms otherthan coagulase-negative staphylococci, is

predictive of CVC infection (79). In pa-tients with nontunneled CVCs, the pres-ence of 50 cfu of organisms by quanti-tative skin site cultures at the time of removal for suspected infection had asensitivity of 75%, a positive predictive value of 100% and a negative predictive value of 92% in detecting CVC infection(84). In another study, a site culture yielding 50 cfu coagulase-negativestaphylococci was associated with a 4.4times greater risk of catheter coloniza-

tion (79). However, in one study, the pos-itive predictive value of nonquantitativeinsertion site culture was only 29% (85).Cultures of the skin insertion site should,therefore, be at least done semiquantita-tively, particularly for the interpretationof cultures growing coagulase-negativestaphylococci. Culture of the skin inser-tion site should only be obtained whenthere is a clinical suspicion of infection;

routine surveillance cultures should notbe done in the absence of suspicion, be-cause they are likely to yield false-positiveresults and cause unjustied catheter re-moval (84).

Question: Can CVC infection be identied as a source of sepsis in nonbacteremic patients without catheter removal for culture?

No; Grade E

Recommendation : When a CVC is sus-pected as a source of sepsis in a nonbac-teremic patient, denitive diagnosis re-quires that the CVC should be removedand sent for culture (in selected cases,after catheter exchange over a guidewire). A denite diagnosis of nonbactere-mic catheter-related sepsis requires thatthe catheter proves colonized, that thereis no other apparent source of sepsis, andthat sepsis abates within 48 hrs of cath-eter removal in the absence of specicantibiotic therapy.

Grade E

Rationale: Only a small proportion (10%to 72%, depending on organisms involved) of infected CVCs are associated with bacteremia (77, 78, 86). When crit-ical care patients develop signs of sepsis,even in the absence of bacteremia, re-placement of central venous catheters isfrequently performed. However, usingstandard culture techniques, rates of proven catheter infection range from8.9% to 26% (87–89), and so many morecatheters are removed than are infected(71, 87, 90). The decision of whether to

remove a CVC in this setting, whereblood cultures are negative, is essentiallya clinical one and involves weighing therisk of catheter replacement against therisk of leaving in place a potential sourceof infection. To help guide this decision, arange of clinical parameters has beensuggested as possible correlates of cathe-ter infection. A number of individual as-sociations have been identied (summa-rized in Table 4), and these have beenused to make recommendations concern-

ing CVC insertion and care (70). Twostudies have assessed the use of such pa-rameters in guiding clinical judgmentand have failed to show they are of valuein increasing the proportion of removedCVCs that are infected (87, 91). Clinicalcriteria have been incorporated with mi-crobiological data and clinical responseto catheter removal into a scoring system(92), which appears more sensitive and

no less specic than the Hospital Infec-tion Control Practices Advisory Commit-tee diagnostic criteria (93, 94) but, nev-ertheless, requires removal of thecatheter for culture.

An elegant approach to the evaluationof a patient with unexplained sepsis whencatheter infection is a possible candidateand there is no local infection or signs of severe sepsis is to replace the catheterover a guidewire. While this practice canbe recommended only in selected casesand is associated with a slightly increased

risk of infection of the second catheter(95), it allows retention of the central venous access and proving or disprovinginfection of the rst catheter. If the rstcatheter proves colonized, the secondcatheter should be removed and anothercatheter inserted at a new site (80).

Question: What strategy should be used to investigate a suspected catheter- related infection?

Grade D

Recommendation : The strategy adoptedto investigate a suspected catheter-related infection (CRI) depends on theclinical severity, the presence of bactere-mia, and the organisms involved. In pa-tients with severe sepsis and suspectedCRI, especially when bacteremia result-ing from one of the “high-risk” organ-isms ( S. aureus , Candida , Pseudomonas )occurs, the catheter should be removed without delay. When a CVC is suspectedas a source of bacteremia, diagnosis of CVC infection may be made by blood cul-

ture-based techniques if: 1) the patient’sclinical condition permits a potentiallyinfected line to be left in place; 2) con-serving the catheter is a primary objec-tive and treatment of CVC infection in situ is considered; or 3) other potentialsources of bacteremia are apparent.Guidewire exchange of the catheter canbe performed in selected cases. While the Acridine Orange Leukocyte Cytospin(AOLC) test offers the possibility of virtu-ally immediate diagnosis, on the basis of




currently available data, its use remainsexperimental.

Grade D

Rationale : The approach to diagnosis andmanagement of suspected CRI has beenreviewed recently (80). The strategyadopted depends on the clinical settingand severity of presentation of the patientand, to some extent, on microbiologicalinformation (i.e., the presence of bactere-mia and microorganisms involved). Incritically ill patients in the ICU with ashort-term CVC suspected of infectionand presenting with sepsis or severe sep-sis, conventional wisdom is to recom-mend catheter removal. This will allowone to make a denite diagnosis of CRI,provided adequate microbiological tech-niques are used, as reviewed above. How-ever, many catheters are removed unnec-essarily following this recommendation.

When a patient who has a CVC in placedevelops bacteremia, the likelihood that

the CVC is the source of the bacteremia will depend on the organism cultured. Inone study of 311 patients who had CVCs,73% of bacteremias were related to CVCinfection, and if the culture was of S. aureus , this gure rose to 92% (77).Other organisms that are particularly as-sociated with CVC infection as a sourceare coagulase-negative staphylococci,Corynebacterium jeikeium , Bacillus spe-cies, and fungi, in particular, Candidaspecies (80).

In patients with suspected CRI and ei-ther 1) a severe sepsis presentation and noother likely source, 2) bacteremia caused by S. aureus , Pseudomonas , or Candida spe-cies, or 3) tunnel infection, or frank inam-mation or purulence at the catheter inser-tion site, the catheter should be removedand cultured (71, 80). Comparisons of cul-tures from blood, catheter site when appli-cable, and catheter segments will provide adenite diagnosis.

In other circumstances, it is possibleto diagnose CRI while leaving the cathe-ter in place. As an initial approach, cath-eter exit-site cultures are useful to ex-clude infection, but their positivepredictive value is low. In some patients with suspected nonbacteremic catheter-related sepsis or coagulase-negativestaphylococcal bacteremia from an un-known source, catheter exchange over aguidewire can be performed in the ab-sence of local inammation (80). In pa-tients with bacteremia and a suspectedcatheter as the source, two approaches to

diagnosing CVC infection without cathe-ter removal for culture, have been re-ported in the literature: cultures of CVCand peripheral blood and the AOLC test,for a rapid diagnosis.

Comparative Cultures of Central Venous Catheter and PeripheralBlood

These approaches are based on the factthat the concentration of bacteria drawn

through an infected catheter is betweenfour (96) and 30 (97) times higher thanthe concentration in peripheral blooddrawn simultaneously. This approach toclinical diagnosis has been assessed intwo ways: 1) by quantitative or semiquan-titative paired blood cultures; 2) by mea-suring time to culture positivity in con-tinuous monitoring blood culturesystems. Both methods rely on it beingpossible to aspirate at least 10 mL of blood through the suspected catheter, which may not be always feasible (98).Furthermore, being culture-based meth-ods, both involve a delay of 24–48 hrsbefore culture results are available, atime interval when a potentially infectedcatheter remains in situ . Their applicabil-ity to the context of the critically ill ICUpatients has not been well assessed.

Quantitative Blood Cultures

A series of articles in the 1980s dem-onstrated that quantitative cultures of

central and peripheral blood can be usedto diagnose catheter-related bacteremia(99–101). Several studies have attemptedto dene the ratio of colony formingunits per milliliter that gives optimal sen-sitivity and specicity. Capdevila et al.(96) found that by using a cutoff of 1:4 forthe ratio of bacterial colonies in periph-eral to central blood, sensitivity and spec-icity of 94% and 100%, respectively,could be achieved. Quilici et al. (102)used a ratio of 1:8 and found a sensitivity

Table 4. Major risk factors for central venous catheter infection

Reference Nos.

Catheter site Subclavian lines are associated with signicantly less catheter site colonization andinfection than jugular or femoral catheters, although less catheter-related bacteremiahas not been demonstrated

74, 88, 117–119

Catheter insertion Difcult catheter insertions with multiple attempts are associated with higher rates of infection

120

Catheter type Multilumen catheters may be associated with a higher risk of infection than single-lumen catheters; this effect is not apparent in RCT performed in critical carepatients; tunneled lines are associated with delayed and fewer incidences of CRB

89, 117, 121; 122–124

Antiseptic-impregnated andantibiotic-coated catheters

Antimicrobial-impregnated catheters are associated with rates of colonization, localinfection, and CRB reduced by about 50% in short-term CVC; when CVCs are usedfor parenteral nutrition this benet may be lost

125–131; 132, 133

Catheter use The use of CVCs for parenteral nutrition has been associated with increasedcolonization and infection rates

117, 134

Duration of catheterization The risk of a central venous catheter being infected increases as the duration of catheterization increases beyond 1 wk

117, 135, 136

Previous catheters Meta-analysis data suggest that “guidewire” replacement of CVC may be associated witha higher infection rate than replacement at a new site; the infection rate for apatient’s rst CVC is signicantly lower than for any subsequent catheter whetherreplaced by guidewire or at a distant site

95; 137

Underlying disease Trauma and burns patients at high risk, neurosurgical patients at lowest risk 88

RCT, randomized clinical trials; CRB, catheter-related bacteremia; CVC, central venous catheter.




of 92.8% and a specicity of 100% in aprospective study of critical care patients.Published expert opinion is that quanti-tative culture of central and peripheralblood samples showing a 5:1 ratio sug-gest CRB (68, 71, 80). A meta-analysis of different catheter culture techniquesconcluded that quantitative blood cul-tures were more cost effective than anyculture technique involving the catheter

itself for diagnosing infection (76).

Continuous Blood CultureMonitoring

This approach is derived from the pre-ceding and takes advantage of the auto-mated blood culture systems (e.g., BacT/ Alert or BACTEC) to detect the timetaken for a blood culture to become pos-itive, which is related to the number of microorganisms initially present: thehigher the initial concentration of anygiven organism, the faster the cutoff point set to determine positivity will bereached (103, 104). Several clinical stud-ies have been performed using pairedcentral and peripheral blood samples andone of the commercially available contin-uous monitoring blood culture systemsthat uses a colorimetric C O2 sensor (105);their ndings are summarized in Table 5(103, 104, 106–110). A differential timeto positivity of 2 hrs favoring the cath-eter blood strongly suggests CRB. Whilethe experience with this technique is in-creasing and appears favorable in cancerpatients and those receiving long-termcatheters, its routine applicability to gen-eral ICU patients remains uncertain. Onlyone study focusing on this population hasbeen published, and its results are notencouraging (110). Further experience isneeded in the ICU population. While its

applicability is probably limited, it isprobably most useful in stable patientshaving bacteremia with a common skincontaminant (e.g., coagulase-negativestaphylococci) and no apparent sourceother than a CVC, a common occurrencein ICU patients.

The Acridine Orange LeukocyteCytospin Test

This technique, described by onegroup in Leeds, derives from a previousone, using cultures of a wire brush passeddown the lumen of a potentially infectedcatheter. In two studies of 115 CVCs ingeneral surgical patients receiving paren-teral nutrition (111) and 22 surgical pa-tients subsequently diagnosed with cath-eter-related bacteremia (90), the authorsfound that these cultures correlated well with a subsequent culture of the cathetertip. To obtain a rapid diagnostic test forCRB while keeping the catheter in place,this group developed the AOLC test, which detec ts bacte ria aspirated orbrushed out from the catheter. The testrequires only 50 L of blood to be with-drawn from a CVC and takes approxi-mately 30 mins and minimal specialistlaboratory expertise. We are aware of fourclinical trials in adults (Table 6). Twohave yielded positive results (112, 113),both from the same group in Leeds, whiletwo from elsewhere (87, 114) both pro-duced disappointing results. None havespecically investigated sepsis patients,and only one specically involved criticalcare patients (114). A further recentstudy from the Leeds group concludesthat the AOLC test is effective and cost-saving (111). At present, given the limitedexperience with this technique and its

conicting results, the technique re-mains experimental.

Community-Acquired Pneumonia

Question: What diagnostic strategy is appropriate in severe community-acquired pneumonia?

Recommendation : Blood cultures, respi-ratory secretions (sputum or endotra-

cheal aspirates) must be obtained. Uri-nary antigen detection of Legionellaserogroup 1 is recommended in endemicareas or during outbreaks. Pleural uidfor Gram-negative stain and cultureshould be obtained if available. Serologymay provide evidence of microbial etiol-ogy in some cases. Invasive proceduresare optional and should be restricted tointubated patients. A diagnostic algo-rithm is proposed.

Grade D

Rationale : Community-acquired pneu-monia (CAP) is a common illness, with anestimated incidence of 2–12 cases/1,000population per year. Most of these casesare successfully managed on an outpa-tient basis; however, 20% will still re-quire hospital admission. Severe CAP isconsidered a distinct clinical entity thatusually requires ICU management andhas a particular epidemiology and asomewhat different distribution of etio-logical pathogens compared with theother less severe forms of community-acquired pneumonia. Severe CAP mayrepresent 10% of the total admissions of aspecialized ICU (138), and the mortalityof these patients is also high. A meta-analysis performed by Fine et al. (139)found a mortality rate of 36.5% in ICU-admitted CAP patients, with a range of 21.7% to 57.3%.

Table 5. Studies of continuous blood culture-monitoring instruments

Population Finding Reference Nos.

Retrospective review of seven patients withsuspected catheter-related infection

Time to positivity shorter for central catheter cultures than culturesfrom peripheral sites

104

Retrospective study of 11 patients in whom CRB was diagnosed

Time to culture positivity 1–24 hrs earlier for central peripheral cathetercultures

106

Retrospective analysis of 64 cancer patients withsuspected CRB

28 cases of CRB were identied; a cutoff of 120 mins had 100%specicity and 96.4% sensitivity

103

Prospective study of 93 cancer patients in an ICU 28 cases of CRB were identied; a cutoff of 120 mins had 91%specicity and 94% sensitivity

107

Prospective study of 269 infectious episodes inimmunocompromised children

31 episodes of bacteremia, 9 CRB, and 24 others; a cutoff of 120 minshad 89% sensitivity and 100% specicity

108

Prospective study in 100 adult general ICU withsuspected CRI

3 patients had CRB, 9 other sources, and 5 nonbacteremic sepsis; nodifference between CRB and non-CRB episodes (2.1 vs. 3.3 hrs)

110

CRB, catheter-related bacteremia; ICU, intensive care unit; CRI, catheter-related infection.




There is not a clearcut denition forsevere community-acquired pneumonia.However, the recent ATS guidelines onCAP (140) dened severe pneumonia

when one of the two major or two of thethree minor criteria were present. Onepractical denition is to regard severeCAP as those patients who require admis-sion to the ICU, but this may result in aselection bias.

Taking into account the potential evo-lution of this disease, prompt institutionof antimicrobial therapy is mandatory,even before any information regardingthe microbiological diagnosis is available.However, obtaining a microbiological di-agnosis is necessary to direct therapy, to

optimize antibiotic treatment, and toavoid the unnecessary use of antibiotics.Most of our knowledge on the diagno-

sis of severe CAP comes from global stud-ies on hospitalized CAP patients. CAPshould be clinically suspected in patients with chest radiograph inltrates with anacute clinical picture that includes one of the following: fever or one respiratorysymptom (cough, expectoration, dyspnea,pleural pain). In an elderly population orin patients with comorbidities, the clini-

cal picture may not be evident in 30% of patients (141).

A microbiological diagnosis of severeCAP is achieved in approximately 50% of

cases (142). As a general rule all samplesfor bacterial cultures must be obtainedbefore the administration of any antibiot-ics. It is recommended that two samplesof blood for bacterial culture be obtained(140, 143, 144). However, a recent studyquestioned the usefulness of blood cul-tures in the routine management of hos-pitalized CAP patients (145). The overallsensitivity of blood cultures in CAP isapproximately 20%.

In the nonventilated patient, a sample of expectorated sputum should be obtained

(140, 143). Overall sputum is available inonly 50% of patients (146). Only 30% of these sputum samples will show criteria forgood quality and will justify processing. Mi-croscopic examination of expectorated spu-tum is the easiest and most rapidly avail-able method of evaluating the microbiologyof lower respiratory tract infections. In hos-pitalized persons with community-acquiredpneumonia, the sensitivity and specicityof sputum Gram stains are 50% to 60% and

80%, respectively, for pneumococcal

pneumonia (147). Specic uorescent an-tibodies can be used to help evaluate spu-tum or other respiratory tract specimensfor the presence of Legionella . Commer-cially available enzyme immunoassay testsare also available for the detection of inu-enza virus, respiratory syncytial virus, ade-novirus, and parainuenza viruses 1, 2, and3. Finally, deoxyribonucleic acid (DNA) am-plication and probe hybridization maypermit rapid detection or identication of

specic microorganisms in clinical speci-mens. These tests are especially appropriatefor recognition of organisms that are fas-tidious or difcult to grow (148). Commer-cial probes for detection of Legionella , M. pneumoniae , and C. pneumoniae are avail-able in some countries.

Bacterial cultures of expectorated spu-tum are not available until 24 hrs. Overallthey are less sensitive than Gram stainsbut more specic. The clear predomi-nance in culture of a potential pathogenicmicroorganism in a valid sample of spu-

tum should be considered diagnostic. Theequivalent of sputum in intubated pa-tients is endotracheal aspirates. Most of the comments mentioned above for spu-tum are valid for Gram stains and cul-tures of endotracheal aspirates. Qualita-tive cultures may be easily contaminatedby pathogens that colonize intubated pa-tients. Quantitative cultures may help todistinguish colonization from infection,but the exact threshold of bacterialcounts to distinguish colonization frominfection has not been validated in severe

CAP.Of great interest in the group of rapiddiagnostic techniques is the detection of urinary antigens. They are usually immu-nochromatographic assays that detectcapsular polysaccharide antigens of S. pneumoniae or Legionella pneumophilaserogroup 1 (149, 150). They can be per-formed in 1 hr, but the diagnostic valueimproves if used on centrifuged urine, which requires more time. As regards L. pneumophila (serogroup 1), the sensitiv-

Algorithm for microbiological diagnosis in severe community-acquired pneumonia

Step 1: Obligatory tests insevere CAP

Two sets of blood cultures

Valid sputum sample for Gram and acid-fast stains and bacterialcultures

Endotracheal aspirates in ventilated patients for Gram and acid-fast stains and cultures

Urinary antigen for Legionella pneumophila in endemic areas orduring outbreaks

Step 2: Optional tests a Urinary antigen for S. pneumoniaeSerologies for atypicals, Legionella species and virusesDNA amplication (PCR) for M. pneumoniae , Legionella , S.

pneumoniae , and viruses. Transthoracic lung punctures innonventilated patientsFiberoptic bronchoscopy with protected specimen brush and/or

BAL

PCR, polymerase chain reaction; BAL, bronchoalveolar lavage. a Optional tests are recommended in individual cases or when the tests from the rst step are

negative or inconclusive.

Table 6. Studies of Acridine Orange Leukocyte Cytospin (AOLC) in adult patients

Setting Finding Reference Nos.

100 patients with suspected CRB, most receivingparenteral nutrition

35 cases of CRB, AOLC positive in 2/17; when used in conjunction withendoluminal brushing, AOLC identied 15/18 and was 100% specic

112

55 CVC tips from ICU patients 4 cases of CRB, AOLC positive in 2; 10 colonized catheters, AOLC positive in 2 11449 patients with suspected catheter-related

bloodstream infection; mixed ICU/HDU12 cases of catheter-related bloodstream infection identied, AOLC negative in all 87

128 surgical patients with suspected CRB 50 cases of CRB, AOLC was 96% sensitive and 92% specic 113

CRB, catheter-related bacteremia; ICU, intensive care unit; HDU, hemodialysis unit.




ity of this technique is approximately70% and the specicity 100%. There isless published experience with S. pneumoniae urinary antigen detection, butthe rst reports are very optimistic withregard to its diagnostic value (150).

In lower respiratory tract infectionscaused by pathogens not readily culturable,serologic techniques may be the sole meansof establishing a diagnosis. Cold agglutinins

are present at a titer of 1:32 (the level re-quired for a rapid bedside agglutinin test) orgreater in more than half of the cases of M. pneumoniae , but the specicity is poor (151).Complement-xing antibodies are specic,but the test requires serial specimens to iden-tify a rise in titer. For C. pneumoniae infec-tion, immunoglobulin M antibodies may notappear for up to 3 wks. In regard to thediagnosis of Legionnaires disease, a positivetiter of 1:256 has a positive predictive value of only 15%, and acute antibodies are usually of low titer. Viruses may cause up 20% of casesof community-acquired pneumonia. Sero-logic tests may be used to diagnose respira-tory viruses, such as inuenza A and B, para-inuenza, adenovirus, and respiratorysyncytial virus (152).

As the microbiological diagnosis of pneumonia is often limited by problemsencountered with the use of expectoratedsputum, in some cases of severe CAP, itmay be necessary to perform an invasiveprocedure to obtain suitable material formicroscopy and culture. The indications forusing invasive techniques in CAP shouldprobably be restricted to patients not re-sponding to initial antibiotic treatment.There is no clear information about thecost effectiveness of using invasive proce-dures in CAP, and in addition, the pub-lished evidence is scanty (153, 154). Trans-thoracic lung puncture cannot beperformed in patients with mechanical ven-tilation because of the subsequent risk of barotrauma. In nonventilated patients, thesensitivity is 40% and the specicity 90%(155). In ventilated patients, beropticbronchoscopy with protected specimenbrush (PSB) or bronchoalveolar lavage

(BAL) are the preferred techniques. Thereis little information as regards the diagnos-tic value of quantitative cultures of PSB orBAL in severe CAP (153, 154).

Algorithm for MicrobiologicalDiagnosis in SevereCommunity-Acquired Pneumonia

Question: Which diagnostic tests are recommended in nonresponding severeCAP?

Recommendation : Up to 10% of patients with CAP do not respond to initial anti-biotic therapy. Mortality in this popula-tion is very high (40% to 50%). A com-plete microbiological reevaluation isrecommended (Table 7). Invasive tech-niques including bronchoscopy with pro-tected specimen brush and bronchoalveo-lar lavage should be performed. Chestcomputed tomographic (CT) scans may

be useful in some patients.Grade D

Rationale : The term nonrespondingpneumonia is used to dene a clinicalsituation in which no adequate responseis achieved, despite appropriate antibiotictreatment for pneumonia (156). Differentterms may be found in the literature, which inc lude nonresolv ing, nonre-sponding, recurrent pneumonia, and pro-gressive pneumonia; it is possible thatthese different terms neither mean norrepresent the same clinical situation forthe different authors who use them. A rational proposal to dene lack of re-sponse is to classify this clinical conceptinto three categories: nonresponding,progressive, and nonresolving pneumo-nia. The rst case would refer to the ab-sence of clinical response to antibiotictreatment (72 hrs–5 days), and the sec-ond would also include an increase inradiographic changes with clinical dete-rioration (rst 72 hrs). Nonresolvingpneumonia would be considered whenthere is persistence in radiographicchanges (at 30 days) after the initial clin-ical response (157). Dening stability isimportant for denitions of nonresponse.

Halm et al. (158) found the median timeto stability of vital signs to be 3 days. Thecriteria used to dene clinical stabilityincluded temperature (37.2°C), heart rate( 100 beats/min), systolic blood pressure( 90 mm Hg), respiratory rate ( 24breaths/min), and oxygen saturation( 90%). The latest ATS guidelines (159)propose three periods in clinical responsethat may orient the clinician in regard to

the evaluation of therapeutic response:the rst on initiation of treatment, thesecond beginning on day 3 when the pa-tient is expected to achieve clinical sta-bility, and the third period being the re-covery from and resolution of theprevious alterations.

The incidence of nonresponding pneu-monia has not been completely estab-lished. Approximately 10% of hospital-ized patients do not respond adequatelyto empirical treatment and another 6%may evolve to progressive pneumonia(157). In a group of patients with nonre-solving pneumonia in CAP, Arancibia etal. (157) observed that 39% evolved toprogressive pneumonia. The mortality of patients with CAP and nonrespondingpneumonia is approximately 40% (157,160), which is three-fold greater than theglobal mortality of patients hospitalizedin a conventional hospital ward and theICU. The probability of death also de-pends on the cause of therapeutic failure.Thus, in nonresponding CAP, the mortal-ity is very high (88%) when the cause is anosocomial infection, being higher thanin primary (38%) or persistent infections(40%) or when diagnosis is not achieved(27%) (157). Causes of nonresponding

Table 7. Microbiological studies indicated in nonresponding community-acquired pneumonia

Sputum Gram and conventional bacterial cultures Legionella IFNormal and modied acid fast stains

Blood cultures 2 setsUrine Capsular antigen for LegionellaBAL Gram and intracellular bacteria

Bacterial cultures and colony countsNormal and modied acid fast stainsGiemsa stainingFungal stains (PAS, silver, etc.) Legionella IF

Bronchial brushing Gram and intracellular bacteriaBacterial cultures and colony countsNormal and modied acid fast stainsGiemsa stainingFungal stains (PAS, silver, etc.) Legionella IF

Pleura Cultures for anaerobesBacterial cultures and colony countsNormal and modied acid fast stains

IF, immunouorescence; BAL, bronchoalveolar lavage; PAS, periodic acid-Schiff.




pneumonia are divided into infectiousand noninfectious (Table 8).

Infectious Causes. Contrary to otherinfections such as meningitis, in pneumo-nia, penicillin resistance in S. pneumoniaedoes not produce excess mortality if theminimal inhibitory concentration (MIC) of penicillin is 4 g/mL and only a smallpercentage of patients (9%) have resistanceto third-generation cephalosporins (161).

However, if the MIC of penicillin for S. pneumoniae is 8 g/mL, therapeutic fail-ure and a greater mortality may be ob-served. Nonetheless, isolated cases of ther-apeutic failure have been described due toresistance to the new uoroquinolones,specically, levooxacin (162), and alsomacrolides (163), making surveillance of the patterns of resistance of S. pneumoniaemandatory.

The presence of infrequent or unusualmicroorganisms is a cause of nonre-sponding pneumonia because these mi-croorganisms are often not adequatelycovered by the empirical therapeutic reg-imens. Within the group of unusual mi-croorganisms are included mycobacteria, Nocardia species (164), anaerobes, lepto-spires, endemic fungi, and P. carinii . Pseudomonas aeruginosa may be thecause of 10% of cases of nonrespondingpneumonia whether due to persistent in-fection or subsequent nosocomial super-

infection (157). In an elderly nursinghome population hospitalized because of CAP, El Sohl et al. (165) found 33% of methicillin-resistant S. aureus , 24% en-teric Gram-negative bacilli, and 14% of P. aeruginosa in cases of therapeutic failureafter 72 hrs of antibiotic administration.The risk factors for CAP caused by P. aeruginosa and other Gram-negative ba-cilli have recently been described: pulmo-

nary comorbidity and previous hospitaladmission (166), and in the ATS guide-lines published in 2001, patients arestratied for those factors to select theinitial treatment (159). Infectious com-plications with local progression or met-astatic infections may produce a slowerresolution or progression of CAP with theappearance of shock, respiratory distress,or multiple organ failure. The incidenceof metastatic infections, such as endocar-ditis, arthritis, or peritonitis, may begreater in bacteremic CAP. Last, in 30%of the cases of nonresponse, no speciccause for lack of response is identied,despite adequate antibiotic treatment.

Noninfectious Causes. The frequencyof noninfectious etiologies in nonre-sponding pneumonia in recent studies isapproximately 10% (160). Table 8 is asummary of the main causes of nonre-sponse in community-acquired pneumo-nia.

In patients with nonresponding or pro-gressive pneumonia, complete clinical re-evaluation is required. The microbiologicalinvestigation should include studies of noninvasive samples, such as sputum, tosearch for infrequent and/or resistant mi-croorganisms, urinary antigen detection, apair of blood cultures, and serum antibodydetection. More recently introduced tech-niques such as polymerase chain reaction

(PCR) in blood and urine may identify S. pneumoniae , Legionella , C. pneumoniae ,and M. pneumoniae in pharyngeal swab(148), although their use is still under development.

Invasive techniques are recommendedin most cases of nonresponding pneumo-nia depending on the clinical situation of the patient. The diagnostic yield is ap-proximately 40% in the context of non-responding pneumonia (153). It is rec-ommended that PSB and BAL becombined in the same procedure (154).Quantitative bacterial cultures of thesesamples are preferred and help to guidethe antibiotic response. Gram stainingperformed in cytocentrifuged BAL uid isalso useful for identifying causal micro-organisms and has predictive value forbacterial growth. Intracellular organismsin polymorphonuclear leukocytes andGram staining in cytocentrifuged BALuid to detect intracellular microorgan-isms are rapidly available and may guidethe initial decision regarding changes inantimicrobial therapy or the differentialdiagnosis of other noninfectious diseases.Microbiological methods should be di-rected toward conventional bacteria, No-cardia , fungi, and opportunistic bacteria.Studies for Legionella should be made with direct immunouorescence and cul-ture or urinary antigen. Recent PCRtechniques with greater sensitivity for thedetection of microorganisms may in-crease the diagnostic yield, although theyshould be carefully interpreted preciselybecause of their capacity to detect molec-ular components or nonviable microor-ganisms. Pleural effusion is a frequent

cause of the absence of a response inpneumonia (160). Table 7 shows a sum-mary of the recommended techniques innonresponding CAP patients.

Imaging Studies. In nonresolvingpneumonia, a simple chest radiographmay demonstrate pleural effusion, the ap-pearance of cavitation, and/or new inl-trates. These ndings are more evidenton a thoracic CT scan, which provides amore detailed study of the parenchyma,the interstitium, the pleura, and the me-

Table 8. Causes of nonresponding pneumonia in community-acquired pneumonia (CAP)

A infectious Resistant microorganisms (CAP) S. pneumoniae S. aureus

Microorganisms not covered by initial therapy P. aeruginosa

Nosocomial infection Acinetobacter MRSA P. aeruginosa

Infrequent microorganisms M. tuberculosis Nocardia speciesFungal pneumonia P. carinii Others

Complications of pneumoniaEmpyema AbscessNecrotizing pneumoniaSeptic metastases

B noninfectious Pulmonary embolism with infarctionNeoplasmsPulmonary hemorrhageEosinophilic pneumoniaPulmonary edema Acute respiratory distress syndromeBronchiolitis obliterans with organizing pneumoniaDrug-induced pulmonary inltrate

Pulmonary vasculitis

MRSA, methicillin-resistant S. aureus .




diastinum. In progressive pneumonia,clinical deterioration and extension of theradiographic changes may appear even within 72 hrs of the initiation of treat-ment (167). Pulmonary CT ndings maydirect the clinician to consider certainmicroorganisms, although radiographicimages are not pathognomonic (167).The appearance of nodular images withthe “halo sign” is suggestive of pulmo-

nary aspergillosis and/or Mucor species(Zygomycetes). Nodules of similar ap-pearance have also been described inCandida , cytomegalovirus, Wegener’sgranulomatosis, Kaposi sarcoma, andhemorrhagic metastasis. P. carinii pneu-monia typically presents as characteristicground-glass opacity or with features of an interstitial pneumonia. Nocardia spe-cies, Mycobacterium tuberculosis , or Qfever may cause bacterial infections withnodules or multiple masses, with or with-out cavitation. Diffuse or mixed intersti-tial inltrates may be due to viruses, suchas inuenza A, respiratory syncytial virus,or M. pneumoniae.

The possibility of differentiating ordiscriminating between infectious andnoninfectious origin by imaging studiesis a currently ongoing eld of study. In astudy on the utility of high-resolution CTscan in acute parenchymatous pulmo-nary disease, Tomiyama et al. (168) foundthat with this technique, correct classi-cation of infectious or noninfectious eti-ology was achieved in 90% of the subjects.

Other imaging studies, such as perfu-sion-ventilation scintigraphy, may be ob-tained to exclude pulmonary embolism, which should be suspected in the absenceof ndings of microorganisms and pa-tients with risk factors, such as postsur-gical recovery, prolonged immobiliza-tion, or signs of deep vein thrombosis,accompanied by respiratory insufciency,hemodynamic instability, and dilation oroverloading of the right ventricle. HelicalCT scan and pulmonary arteriographycomplement this diagnosis.

Ventilator AssociatedPneumonia

Question: What are the clinical signs and symptoms indicative of VAP as a cause of severe sepsis?

New pulmonary inltrates plus two of the following: fever 38°C, leukopenia,leukocytosis, purulent secretions, or aclinical pulmonary infection score (CPIS)

6.

Grade D

Recommendations :

1. The presence of new chest radiographinltrates plus two of the three clini-cal variables (fever 38°C, leukocyto-sis or leukopenia, and purulent secre-tions) is useful for the clinicalscreening of VAP (high sensitivity).The modied clinical pulmonary in-

fection score ( 6) is also useful forinitial screening.2. However, for patients suffering from

acute respiratory distress syndromeand for whom it is difcult to demon-strate deterioration of radiologic im-ages, at least one of the three preced-ing may sufce to activate initialscreening. A high index of suspicionshould also be maintained in cases of unexplained hemodynamic instabilityor deterioration of blood gases in pa-tients requiring mechanical ventila-

tion.3. The combination of the presence of pulmonary inltrates plus two of thethree clinical signs is sufcient to con-sider initiating antibiotic treatment. A modied CPIS of 6 can be also usedfor this purpose. In both cases, addi-tional procedures are needed, such ascultures of lower respiratory tract se-cretions, before starting therapy.

Grade D

Rationale : The rst step in the process of

assessing suspected VAP is to identifyclinical signs and symptoms. The tradi-tional clinical criteria of fever, leukocyto-sis, and purulent tracheal secretions,usually including the presence of radio-graphic inltrates, are the initial indica-tors of hospital-acquired pneumonia(HAP) in ventilated and nonventilated pa-tients (169). The accuracy of clinical di-agnosis of VAP has been investigated inseveral studies (169), which taken to-gether indicate that the diagnostic crite-ria of a radiographic inltrate and one

clinical feature (fever, leukocytosis, orpurulent tracheal secretions) have a highsensitivity, but the specicity is low. In astudy in which the “gold standard” washistology plus a positive microbiology of immediate postmortem lung samples,the presence of chest inltrates plus twoof three clinical criteria resulted in a 69%sensitivity and 75% specicity (170).

An attempt to solve the problem of thelow specicity of using a single criterionplus radiographic inltrates was made by

Pugin et al. (171) some years ago. Theseauthors combined clinical, radiographic,physiologic (Pa O2 /FIO2 ), and microbiolog-ical variables (semiquantitative culturesof endotracheal aspirates). When CPIS was 6, there was a good association with the presence of pneumonia denedby bacterial index of the BAL culture. Theproblem with this score is that it is notuseful for the initial screening of VAP

because it needs some microbiological in-formation that may take some time to beavailable. Singh and colleagues (172)used a modied CPIS (using a Gramstain) for clinical treatment decisions.Using histology plus immediate postmor-tem lung quantitative lung cultures as areference standard, the CPIS resulted in asensitivity of 77% and a specicity of 42%in another study (170). A very recentstudy validated the modied CPIS in apopulation of mechanically ventilated pa-tients, nding a 60% sensitivity and a59% specicity (173).

For patients suffering from acute re-spiratory distress syndrome and for whom it is difcult to demonstrate dete-rioration of radiologic images, at leastone of the three clinical criteria describedabove may sufce to activate an initialscreening (174). A high index of suspi-cion should also be maintained in cases of unexplained hemodynamic instability ordeterioration of blood gases in patientsrequiring mechanical ventilation. In theabsence of any of these ndings, no fur-ther investigations are required and ob-servation will sufce.

Question: What is the preferred specimen for lower respiratory secretions in VAP?

Quantitative or semiquantitative culturesof endotracheal aspirates, blind or bron-choscopic PSB, or BAL samples. The de-tection of intracellular organisms ( 5%)in BAL uid is a very specic marker for VAP.

Grade B

Recommendations : Quantitative or semi-quantitative cultures of nonbroncho-scopic (endotracheal aspirates or “blind”PSB or BAL) or bronchoscopic tech-niques are preferred (bronchoscopic PSBand BAL). Because these tests have sim-ilar sensitivities, specicities, predictive values, and likelihood ratios, the choicedepends on local expertise, experience,availability, and cost factors. The ndingof 5% of intracellular microorganisms




in centrifuged BAL uid is a highly spe-cic marker of VAP.

Grade B

Rationale : The logical step after clinicalsuspicion of VAP has been established istrying to obtain a microbiological diag-nosis with the aim of conrming VAP andto adjust the initial antimicrobial treat-ment. The information provided by the

sampling techniques may serve for bothpurposes. Several sampling techniqueshave been proposed and investigated inobservational studies. Overall, it is dif-cult to compare studies due to the heter-ogeneity of the populations included, thereference gold standard used, and the useof prior antibiotics before obtaining sam-ples. Even the denition of what is priorantibiotic treatment is not consistent when comparing studies (175–177).There are two problems when evaluatingdiagnostic tests for VAP. First, prior an-

tibiotic treatment clearly modies thesensitivity of cultures (178), and second,there is a variety of reference tests used inthe studies that make comparisons verydifcult. In fact, the only absolutely ac-curate reference test should be the com-bination of immediate postmortem lunghistology and microbiology, a gold stan-dard used in few of the studies (170, 179).Most of the recommendations mentionedbelow are supported by two recent con-sensus conferences: The European TaskForce on VAP (180) and the ConsensusConference on ICU-Acquired Pneumonia(181).

The problem of the gold standard isinherent to most of the studies on VAPbecause there is not a clearcut referencestandard. However, those studies thatused histology and or microbiology of immediately obtained postmortem lungsamples have to be regarded as optimal.The main conclusions are:

1. VAP is a multifocal process that mostfrequently involves the lower and pos-terior segments of the lungs bilater-

ally, in which different phases of severity and evolution coexist. Lungdamage is found very frequently andcoexists with different stages of pneu-monia.

2. The relationship between histologyand quantitative cultures is highlycomplex, frequently complicated bythe presence of preexisting lung dam-age. In some cases, quantitative cul-tures of lung tissue cannot discrimi-nate between the presence and

absence of histologic pneumonia andthey are independent of the evolutionof VAP.

3. Prior antibiotic treatment has an im-portant inuence on the lung bacte-rial burden and, consequently, inu-ences the negativity of quantitativecultures, depending on the thresholdsused.

4. Endotracheal aspirates (EA), BAL, and

PSB samples are effective at identify-ing causative microorganisms of VAPin these postmortem studies. BlindBAL and PSB methods are equally ef-fective as conventional bronchoscopicprocedures.Blood cultures are the rst step in the

microbiological diagnosis of VAP. Overallthe sensitivity of the method does notexceed 25%. In addition, the specicitymay vary because other extrapulmonarysources may be responsible, particularlyin mechanically ventilated patients. Agreement between blood cultures andcultures of respiratory secretions con-rms the microbial causality.

EA are the simplest noninvasivemeans of obtaining respiratory secretionsfrom mechanically ventilated patients. Asfor sputum, the assessment of the qualityof the sample is imperative before pro-cessing. From old and more recent liter-ature, it is clear that qualitative culturingof EA is highly nonspecic (175). Quali-tative EA cultures usually identify organ-isms found by invasive tests (high sensi-tivity); however, they have only amoderate positive predictive value. Thenegative predictive value is high in theabsence of prior antibiotic treatment.Some studies have investigated the accu-racy of quantitative cultures of EA in VAP. The cutoff point established was105 –106 cfu/mL. Sensitivity ranged from38% to 82%, and the mean sensitivity was 76%. Specicity ranged from 72% to85%, and the mean specicity was 75%(182). According to these studies, thesensitivity and specicity of quantitativetests on cultures of EA samples varies

widely in their ability to diagnose VAP butoverall the diagnostic value is very rea-sonable and not very different in terms of sensitivity and specicity to that obtainedby invasive samples.

Quantitative cultures of BAL havebeen used for many years in the diagnosisof pneumonia. Because of the initial de-scription of bronchoscopic BAL in VAP,several methodologies have been usedthat include protected and nonprotectedsystems and different volumes injected.

The assessment of the quality of the sam-ples should be done looking at the per-centage of epithelial squamous cells, al-though very few studies have conrmedthe quality of the samples and only onelooked at the reproducibility of the tech-nique, which appeared reasonable (183). An evidence-based study reviewed 23studies on BAL in VAP, all of them pro-spective (184). The most widely used

threshold was 104

cfu/mL. The sensitivityranged from 42% to 93%, and the meansensitivity was 73%. Specicity rangedfrom 45% to 100%, and mean specicity was 82%. The likelihood ratio rangedfrom 0.9 to 11.6. Twelve of these 23 stud-ies looked for intracellular bacteria, witha mean sensitivity of 69% and a meanspecicity of 75%. The threshold for de-tection used ranged from 2% to 5%. Theclinical implication of these results arethat BAL cultures are not diagnostic forpneumonia in almost one fourth of casesand that the diagnosis is incorrect inabout 20% of cases. Detecting intracellu-lar bacteria in BAL culture is a quick andspecic test and has a high predictive value.

The PSB technique has been used foralmost 20 yrs in diagnosing pneumonia,and variations in its methodology havebeen applied to the diagnosis of VAP. Thecutoff point used for quantitative cultures was 103 cfu/mL. The majority of studiesreviewed did not control for the quality of samples. Only two studies looked at thereproducibility of the technique (185,186).

Most of the studies were comparative with other techniques, such as EA and orBAL. An evidence-based review foundthat the sensitivity of the techniqueranged from 33% to 100%, with a meansensitivity of 66% (187). The specicityranged from 50% to 100%, with a meanspecicity of 90%. The median likelihoodratio was 16. Overall, PSB appears morespecic than sensitive in diagnosing VAP,and in patients with suspected VAP and apositive result from a PSB sample, the

likelihood ratio of VAP appears to bemuch 1. The PSB technique is themethod that offers the best sensitivity/ specicity relationship.

Because beroptic bronchoscopy isnot exempt from risks and is inconve-nient, other methods of sampling havebeen developed. The rationale for usingblind methods is based on the multifocalnature of VAP. Overall, three types of blind methods have been reported: blindbronchial sampling, blind BAL (“mini-




BAL”), and blind use of PSB (188). Thesensitivity varied from 74% to 97% forblind bronchial sampling, from 63% to100% for mini-BAL, and from 58% to86% for blind PSB. The specicity variedfrom 74% to 100% for blind bronchialsampling, from 66% to 96% for mini-BAL, and from 71% to 100% for blindPSB. The likelihood ratio for the threetypes of techniques ranged from 2.26 to

18.25. The agreement with beroptictechniques (PSB) varied from 73% to100%. Overall, one can say that there is alack of standardization of the differenttechniques used. The limited number of studies performed to date suggests thatthe sensitivity and specicity of the blindtechniques is similar to that with ber-optic bronchoscopy techniques. Side ef-fects of blind techniques appear to beminimal and, at worst, are similar tothose for bronchoscopic techniques.

Question: What strategy should guide

initial diagnosis and management? A quantitative strategy (Fig. 1) is pre-ferred.

Grade B

A clinical strategy using the criteriamentioned in question 1 is also proposed when quantitative cultures are not avail-able, although this is less specic thanthe quantitative strategy.

Grade B

In both cases, samples should be ob-tained before new antibiotic administra-tion.

Grade D

A negative culture of an endotrachealaspirate sample without prior antibiotictreatment rules out VAP.

Grade D

Recommendation : Available evidencesuggests that reliance on clinical signsand endotracheal aspirate culture resultsleads to overdiagnosis of HAP. When per-formed before the introduction of antibi-otics (or the use of a new regimen in apatient who has previously received anti-biotics), the use of quantitative culture

techniques after having obtained PSBand/or BAL specimens from the lung of patients with signs suggestive of HAP al-lows denition of a therapeutic strategysuperior to that based exclusively on clin-ical evaluation, minimizing the use of unnecessary antibiotics, with no adverseeffects on patient outcome (Fig. 1). Inpatients with clinical evidence of severesepsis or patients with a very high pretestprobability of the disease, the initiation of antibiotic therapy should not be delayedand patients should be treated immedi-ately with broad-spectrum antibiotics,even when no bacteria are detected usingmicroscopic examination of pulmonarysecretions. Although the true impact of this decision tree on patient outcome re-mains controversial, being able to with-hold antimicrobial treatment from somepatients without infection may constitutea distinct advantage in the long term, byminimizing the emergence of resistantmicroorganisms in the ICU and redirect-ing the search for another (the true) in-fection site.

Figure 1. The “quantitative” strategy. *Pulmonary inltrates plus two of three clinical parameters or a clinical pulmonary infection score of 6. ICO,intracellular organisms.




When quantitative culture techniquesare not available in patients clinically sus-pected of having HAP, we recommendfollowing a clinical strategy (Fig. 2), em-phasizing that antimicrobial treatment

should be reevaluated on day 3, whensusceptibility patterns of the microorgan-ism(s) considered to be causative areavailable, to select treatment with a nar-rower spectrum or even to stop antimi-crobial therapy if the clinical picture isbarely compatible with a diagnosis of pneumonia. In both strategies (Figs. 1and 2), samples of respiratory secretionsfor culturing should be obtained beforeantibiotic administration or antibioticchanges for pneumonia to avoid false-negative results. In addition, a negative

culture of respiratory secretions in theabsence of prior antibiotic treatmentrules out bacterial pneumonia in mostcases.

Grade D

Rationale : Ideally, any diagnostic strategyintended to be used in patients clinicallysuspected of having developed VAPshould be able to reach the three follow-ing objectives: 1) to accurately identifypatients with true pulmonary infection

and, in case of infection, to isolate thecausative microorganisms (to initiate im-mediate appropriate antimicrobial treat-ment and then to optimize therapy basedon susceptibility patterns); 2) to identify

patients with extrapulmonary sites of in-fection; and 3) to withhold and/or with-draw antibiotics in patients without in-fection.

Two diagnostic algorithms can beused in cases of suspected VAP. One op-tion is to treat every patient clinicallysuspected of having a pulmonary infec-tion with new antibiotics, based on thefact that several studies have shown thatimmediate initiation of appropriate anti-biotics was associated with reduced mor-tality (189–192). In this option, the se-

lection of appropriate empirical therapyis based on risk factors and local resis-tance patterns and involves qualitativetesting to identify possible pathogens, an-timicrobial therapy being adjusted ac-cording to culture results or clinical re-sponse (Fig. 2). This “clinical” approachhas two potential advantages: rst, nospecialized microbiological techniquesare required, and second, the risk of miss-ing a patient who needs antimicrobialtreatment is minimal, at least when all

suspected patients are treated with newantibiotics. However, such a strategyleads to overestimation of the incidenceof VAP because tracheobronchial coloni-zation and noninfectious processes mim-icking pneumonia are included. Qualita-tive endotracheal aspirate cultures clearlycontribute to the diagnosis of VAP only when they are completely negative for apatient with no modication of prior an-

timicrobial treatment. In such a case, thenegative predictive value is very high andthe probability of the patient havingpneumonia is close to nil (193).

Concern about the inaccuracy of clin-ical approaches to VAP recognition hasled numerous investigators to postulatethat “specialized” diagnostic methods, in-cluding quantitative cultures of endotra-cheal aspirates or specimens obtained with bronchoscopic or nonbronchoscopictechniques including BAL and/or PSB,could improve the identication of pa-tients with true VAP and facilitate deci-sions whether or not to treat, and thusclinical outcome (194–197). By usingsuch a strategy, the decision algorithm issimilar to the one described in Figure 2,except that therapeutic decisions aretaken based on results of direct examina-tion of distal pulmonary samples and re-sults of quantitative cultures.

Other than decision-analysis studies(198, 199) and one retrospective study(200), four trials have so far assessed theimpact of a diagnostic strategy on antibi-otic use and outcome of patients sus-pected of having VAP (195, 201–203). Nodifferences in mortality and morbidity were found when either invasive (PSBand/or BAL) or noninvasive (quantitativeendotracheal aspirate cultures) tech-niques were used to diagnose VAP inthree Spanish randomized studies (201–203). However, those studies were basedon relatively few patients (51, 76, and 88,respectively), and antibiotics were con-tinued in all patients despite negative cul-tures, thereby neutralizing one of the po-tential advantages of any diagnostic test

in patients clinically suspected of having VAP. Concerning the latter, several pro-spective studies have concluded that an-tibiotics can, indeed, be stopped in pa-tients with negative quantitative cultures with no adverse effects on the recurrenceof VAP and mortality (192, 200, 204–206). One of the rst studies to clearlydemonstrate a benet in favor of invasivetechniques was a prospective, random-ized trial that compared the two strate-gies in 413 patients suspected of having

Figure 2. The “clinical” strategy. *Pulmonary inltrates plus two of three clinical parameters or aclinical pulmonary infection score of 6.






Question: Should abdominal uid collec-tions identied by imaging be aspirated as a matter of routine?

Yes; Grade E

Specimens should be obtained forGram stains and aerobic and anaerobiccultures.

Recommendation : Collections identiedby imaging studies should, if possible, beaspirated and drained during imagingand a substantial volume of uid col-lected for rapid Gram staining and forboth aerobic and anaerobic cultures.

Grade E

Rationale : Differentiation of infected ma-terial from hematoma or inammatoryuid is not possible on the basis of imag-ing studies alone (223). Conrmationthat infection is present and identica-tion of possible drug-resistant organisms

relies on obtaining samples for micros-copy and culture (226–229). While infec-tions that develop in clean wounds arefrequently caused by skin ora, including S. aureus , when contaminated or dirty wounds become infected, it is possible toidentify at least one anaerobic organismin 65% to 94% of samples (230). Indeed,

50% of abdominal abscesses arepolymicrobial and almost 80% involve atleast one anaerobic species (221, 231–233). Detection of anaerobic organismsin clinical specimens is technically more

demanding than performance of aerobicbacterial cultures. If anaerobic organismsare to be recovered successfully, specicmeasures need to be taken in obtainingsamples, such as prompt transportationof pus to the microbiology laboratory. Anaerobes grow more slowly than aer-obes, and that coupled with the polymi-crobial nature of such infections oftenmeans that nal culture results may re-quire several days. For that reason, theresults of an initial Gram stain of theuid may provide very useful information

to guide initial therapy (234).Question: Is there value in obtaining ab-dominal drain uid for culture?

No; Grade E

Such specimens may yield misleadingresults due to supercial contaminationof the distal portions of drains. Excessantibiotic use could well result from thereliance on culture data from surgicaldrains.

Question: Are blood cultures recommended routinely with intraabdominal infections?

Yes; Grade E

Recommendation : Two sets of blood cul-tures should be obtained.

Grade E

Rationale : Intraabdominal infections fre-

quently result in bacteremia. Blood cul-tures may reveal the etiology of an intra-abdominal infection before samples fromthe suspected site of infection itself canbe obtained. However, blood culturesmay not reveal the full spectrum of or-ganisms responsible for polymicrobial in-fections, especially the anaerobic compo-nents of such infections.

Question: Is it sometimes necessary to request a quantitative (i.e., determina-tion of the MIC) antimicrobial suscepti-bility test instead of a test that simplyindicates if a bacterial pathogen is sus-ceptible or resistant?

Yes; Grade E

Recommendation: There are very fewclinical situations that have been shownto benet signicantly from the determi-nation of antimicrobial agent MICs ratherthan test methods that indicate that anisolate is either susceptible or resistant. Itis important, however, with a few antibi-otics and organisms to determine an MICto accurately determine whether an or-

ganism is susceptible, e.g., penicillins andextended spectrum cephalosporins withpneumococci. Other than those exam-ples, there is evidence that MIC tests areuseful in patients who have infective en-docarditis and, perhaps, chronic osteo-myelitis to help select the most potentagents for long-term therapy.

Grade E

Rationale : We are presently in an era of emerging antimicrobial resistanceamong several common bacterial patho-

gens that may be associated with sepsis(235–237). This includes methicillin re-sistance in community-acquired as wellas hospital-acquired strains of S. aureus(238), vancomycin-intermediate or -re-sistant strains of S. aureus (239, 240)multiple drug resistance in S. pneumoniae (241, 242), vancomycin resis-tance in enterococci (243), production of extended-spectrum or inducible -lacta-mases that affect the latest generationpenicillins and cephalosporins in some

members of the Enterobacteriaceae(244), and Pseudomonas aeruginosa iso-lates that may be resistant to all com-monly prescribed antibiotics. Strains of these organisms may not respond to em-pirical therapeutic choices due to thepresence of acquired resistance mecha-nisms. Immunocompromised patients with sepsis may be especially likely toharbor one of these highly resistant

pathogens.One of the most important tasks of theclinical microbiology laboratory is theperformance of antimicrobial susceptibil-ity tests on bacterial isolates involved inserious infections, including sepsis. Thegoal of a susceptibility test is to predictthe likely outcome of treating a patient’sinfection with a particular antimicrobialagent. Empirical therapy continues to beeffective for some bacterial pathogens be-cause resistance mechanisms have not yet been acquired or are still very rare,e.g., penicillin is still a very effective ther-apy for Streptococcus pyogenes ; erythro-mycin resistance has not yet occurred in Legionella and Mycoplasma . The two im-portant and related functions of suscep-tibility testing are the detection of frankresistance and the verication of suscep-tibility of isolates to standard therapeuticchoices.

Susceptibility Testing Methods

Among the earliest methods of antimi-crobial susceptibility testing were thebroth tube dilution or agar plate dilutionprocedures. This involved preparing two-fold dilutions of antibiotics in broth oragar bacterial growth media (245)). Theantibiotic-containing tubes or plates werethen inoculated with a standardized bac-terial suspension. Following overnightincubation, the tubes or plates are exam-ined for visible evidence of bacterialgrowth. The lowest concentration of an-tibiotic that prevents visible growth rep-resents the endpoint of the test, the min-imal inhibitory concentration. The

advantages of this technique include thegeneration of a quantitative result (i.e.,the MIC) (246)). The principal disadvan-tages of the methods include the tedious,manual task of preparing the antibioticsolutions for each individual test and thepossibility of making errors in the prep-aration of the antibiotic test concentra-tions. Nevertheless, the agar dilutionmethod is considered the standard refer-ence procedure for antibiotic testing inEurope (247)). While it is possible to




measure the bactericidal effect of an an-tibiotic (the minimal bactericidal concen-tration) by the broth dilution method,there is little evidence that such testing would be important in a septic patient;rather, bactericidal tests may be useful inmonitoring patients with infective endo-carditis or, perhaps, chronic osteomyeli-tis (248, 249).

A modication of the tube dilution

test has made broth dilution testing verypopular in the United States. Miniaturiza-tion and mechanization has been accom-plished by the use of small, disposable,plastic microtiter trays for susceptibilitytesting. These microdilution panels ordi-narily contain 96 wells, each holding a volume of 100 L, which allows approx-imately 12 antibiotics to be tested in arange of eight- to two-fold dilutions insingle plastic trays (250) available fromseveral commercial sources. The advan-tages of the microdilution susceptibility

method include the economy of reagentsand space that occurs due to miniaturiza-tion, the reproducibility of results due topreparation of a large number of traysfrom the same antibiotic dilution series,and the convenience of having pre-prepared panels available wheneverneeded.

Antimicrobial Gradient Method

The E test (AB Biodisk, Solna Sweden)is a commercial product that uses theprinciple of the establishment of an anti-microbial gradient in an agar medium asa means of determining susceptibility(251). The E test consists of thin plastictest strips that are impregnated on theunderside with a dried antibiotic concen-tration gradient and are marked on theupper surface with a concentration scale.E strips containing different antibioticsare placed on the surface of an appropri-ate agar plate that has been inoculated with a standardized bacterial suspension.

After overnight incubation, MICs can bedetermined by viewing the strips fromthe top with the plate lids removed. Anantibiotic gradient formed in the agaradjacent to the E test strips gives rise toan elliptical-shaped inhibitory areaaround the strip. The MIC is determinedby the intersection of the bacterialgrowth ellipse with the concentrationmarked on the E test strip. The E test hasbeen particularly popular for testing fas-tidious or anaerobic bacteria (252, 253).

Disk Diffusion Method

The simplest and one of the mostreliable susceptibility testing methodsis the disk diffusion procedure. The testis performed by applying a standardizedbacterial inoculum to the surface of aspecial (usually Mueller-Hinton) agarplate (254). From 6 to 12 commerciallyprepared lter paper antibiotic diskscan be placed on the agar surface, depend-ing on the size and shape of the plates used.Plates are incubated overnight before mea-surement of the diameters of the zones of growth inhibition around each of the anti-biotic disks. The diameter of the zone of inhibition is related to the susceptibil-ity of the isolate and to the diffusionrate of the drug through the agar me-dium. The zone size is then interpretedusing a table of values developed andpublished by a standards organization,such as the National Committee forClinical Laboratory Standards (NCCLS)in the United States (254) or by one of the national committees in Europe(255). The results of the disk diffusiontest are “qualitative” in that a categoryof susceptibility (i.e., susceptible, inter-mediate, or resistant) is derived fromthe test rather than an MIC (254). Theadvantages of the disk diffusion methodare the simplicity and low cost of thetest and the exibility to select any bat-tery of drugs for testing. Disadvantagesof the disk test are the lack of mecha-nization and the fact that some fastidi-

ous or slow-growing bacteria may notbe accurately tested by that approach.

Short-Incubation AutomatedInstrument Systems

The use of instrumentation can allowperformance of susceptibility tests in ashorter period than manual readings be-cause the use of sensitive optical detec-tion systems can provide measurement of subtle changes in bacterial growth. Thereare two instruments presently cleared by

the Food and Drug Administration foruse in the United States that are capableof generating rapid (4–10 hrs) suscepti-bility test results (256).

The Vitek and Vitek 2 Systems (bio-Merieux Vitek, Durham, NC) use com-pact plastic reagent cards that containminute quantities of antibiotics andtest media in 45- to 64-well formats forantimicrobial susceptibility testing.Both instrument versions use repetitiveturbidimetric monitoring of bacterial

growth during an abbreviated incuba-tion period of approximately 6–10 hrs.The instruments allow testing of com-mon, rapidly growing Gram-positiveand Gram-negative aerobic bacteria, as well as streptococci (256).

The MicroScan WalkAway (Dade Mi-croscan, West Sacramento, CA) is a largeself-contained incubator/reader devicethat can simultaneously incubate and an-

alyze from 40 to 96 standard size mi-crodilution trays (256). The instrumentincubates the panels and examines themperiodically with a photometer to deter-mine growth development. Overnight in-cubation is required with most organ-isms, but early readings of the panels maybe able to detect high-level resistance with some organisms and drugs.

A shortcoming of rapid susceptibilitytesting by the current and previouslymarketed instruments has been some dif-culty in detection of inducible antimi-crobial resistance in a consistent manner(256, 257). In some instances, a shortincubation period of 4– 6 hrs was notsufcient for induction of an enzyme(e.g., some -lactamases) that would de-stroy the antibiotic being tested (258).Thus, the rapid test might indicate sus-ceptibility to an antibiotic, whereas aconventional overnight test would allowtime for enzyme induction resulting inobvious resistance. However, improve-ments have been made through modi-cations of the computer software to ei-ther provide prolonged incubation forproblematic organism-drug combina-tions or by detection of probable errorsusing “expert” software to detect and cor-rect atypical results (256). Moreover,there may be a clinical and economicadvantage to determining antimicrobialsusceptibility in a short period (259, 260).This could be especially useful in patients with sepsis in which there is a bacterialisolate available for rapid testing.

Regardless of which antimicrobial sus-ceptibility method is used, the importantprinciple is that antimicrobial resistancemust be detected quickly and reliably toavoid potential clinical failures. Further-more, it is important to verify the suscep-tibility of a patient’s bacterial isolate tothe drug(s) chosen for empirical therapy.Many physicians consider the susceptibil-ity test information to be equal to ormore important than the identity of spe-cic bacterial pathogens in cases of severe infection.




When Is It Necessary toMeasure Serum Levels of Antibiotics?

Because aminoglycoside nephrotoxic-ity is largely associated with prolongedelevated trough concentrations of thesedrugs, it is important for patient safety(especially in patients with impaired re-nal function) to monitor trough levels of

gentamicin, tobramycin, or amikacin, whether administered using conventionalmultiple-daily dosing regimens or once-daily dosing. It is now recognized thataminoglycosides kill bacteria in a concen-tration-dependent manner and that tox-icity does not seem closely related totransient high drug concentrations (261).Thus, it is not routinely useful to monitor“peak” aminoglycoside blood levels. Like- wise, there is little evidence that vanco-mycin nephrotoxicity or ototoxicity is re-lated to peak blood levels. It may beuseful to determine vancomycin troughconcentrations in patients with signi-cant renal impairment to assure adequateblood levels for efcacy and to avoid pos-sibly toxic prolonged trough levels. Someexperts have found it useful to monitorother drug levels (e.g., trimethoprim-sulfamethoxazole, penicillins, and chlor-amphenicol) in patients with signicantlyimpaired renal or hepatic function, al-though measurement of antibacterialsother than aminoglycosides and vanco-mycin is often not readily available. Itra-conazole capsule absorption is dependenton gastric acidity and may be greatly di-minished in patients receiving antacids,H2 blockers, or proton pump inhibitors.Thus, it is useful to measure itraconazoleblood levels to assure efcacy when thatpreparation is used.

Question: What approaches should betaken to the diagnosis and treatment of invasive fungal infections in ICU pa-tients?

Recommendations : In sepsis patients atrisk of an invasive Candida infection,

clinical examination must include dilatedfunduscopy. The following are indica-tions for systemic antifungal therapy:

● Candidemia● Candida species isolated from a CVC

tip● Isolation of Candida species from any

sterile site except urine● Identication of a yeast on microscopy

from any sterile site pending cultureresults

Rationale : Clinical diagnosis : Clinicalfeatures of invasive Candida infection arein most cases nonspecic, ranging fromunexplained fever to sepsis (262). Specicclinical manifestations are rare. Candidalchorioretinitis occurs in 15% of candi-demic patients (263) but, when found, isan absolute indication for the initiation of antifungal therapy. Skin lesions and sep-tic arthritis occur less frequently still

(264, 265). Microbiological diagnosis : The devel-opment of invasive Candida infectioncorrelates with preceding colonization.Sites screened should include urine, rec-tum, gastric aspirate, vascular accesssites, sputum/throat swab, wounds, andsurgical drains. The number of sites col-onized predicts the risk of invasive infec-tion (266, 267). A cutoff of two sites col-onized, as an indication for beginningempirical antifungal therapy, has a highsensitivity but low specicity, 22% in onestudy (267). Taking the number of colo-nized sites alone as a measure of infectionrisk makes no account of the intensity of colonization. This has been taken intoaccount using semiquantitative culturetechniques to produce a “corrected Can-dida colonization index”; sensitivity andspecicity of 100% was achieved in a ret-rospective analysis of 29 ICU patients(267). No evidence exists to direct howfrequently samples to detect colonizationshould be obtained; 5 days would seem areasonable interval. At present, there isno consensus on the value or use of rou-tine screening for Candida in all ICU pa-tients and the data are inadequate to sup-port a general recommendation that itshould be instituted.

In the past, conventional blood cul-ture techniques were relatively insensi-tive in detecting blood-borne Candida in-fections, although this is less of aproblem with current instruments. Whilegrowth of Candida species in blood is aclear indication for the initiation of anti-fungal agents, failure to prove candi-demia in an at-risk patient in no way

disproves the diagnosis.Candiduria in patients who have nothad instrumentation of the renal tract isstrongly suggestive of renal involvementin disseminated candidiasis. The practical value of this is limited by the fact that themajority of ICU patients will have beencatheterized at some stage. Furthermore,up to 50% of candidemic patients will nothave candiduria (268). In an ICU setting,the nding of candiduria in a catheter-ized patient is no more signicant an

indicator of invasive disease than isola-tion from any other single site (269).

While a single colony of Candida spe-cies isolated from a sterile site, such asblood or cerebrospinal uid, must be re-garded as signicant, the greatest obsta-cle to the diagnosis of invasive Candidainfection by culture from nonsterile sitesis distinguishing infection from coloniza-tion. At certain sites, e.g., central venous

catheter tip, cutoff values in quantitativeculture have been established, which at-tempt to make this distinction ( 15 cfuby the roll plate method; 100 cfu bysonication). Diagnosis of a Candida infec-tion by tissue biopsy is made on the basisof either a quantitative culture of 105

organisms per gram of tissue or the pres-ence of yeasts on microscopy pendingculture results.

Although Candida albicans continuesto make up the majority of clinical iso-lates, the incidence of non- albicans spe-cies is increasing, e.g., Candida glabrata ,Candida parapsilosis , and Candida tropi-calis . When Candida species is culturedfrom nonsterile sites or urine, differenti-ation of C. albicans from non- albicansspecies using the germ-tube technique isgenerally sufcient. However, when Can-dida species is identied at sterile sites,speciation and susceptibility testingshould be performed. The susceptibilityof these species to azole antifungals var-ies. In certain cases, the use of azoles maybe possible in place of amphotericin withits associated toxicity (270). In addition,knowledge of the species and resistancepatterns within a particular ICU is essen-tial to the choice of empirical antibiotics.

Nonculture-Based DiagnosticTechniques

A number of PCR (271) and serologi-cal (272–274) assays have shown promisein preclinical trials, but two trials in ICUpatients found that such techniques areunlikely to replace a culture-based diag-nosis (275, 276).

Question: When is it important to request antifungal susceptibility testing of a Candida isolate from the blood culture of a septic patient?

Antifungal susceptibility testing canbe helpful in immunocompromised pa-tients with C. albicans infections follow-ing prolonged prophylaxis or with ther-apy with one of the azole class drugs,especially uconazole. It may also behelpful to determine the susceptibility of




a C. glabrata or C. tropicalis isolate toone or more of the azoles.

Grade E

Rationale : Antifungal susceptibility test-ing was an arcane pursuit 10 years ago when few antifungal agents were avail-able for therapy, before resistance mech-anisms were appreciated in fungi, andbefore standardization of testing meth-

ods. However, there are now several dif-ferent triazole antifungal agents (e.g., u-conazole, itraconazole, voriconazole,posaconazole), an echinocandin (caspo-fungin), ucytosine, and regular and lipid versions of amphotericin. In addition,certain Candida species are known tohave intrinsic resistance to some agents,e.g., C. krusei to the azoles, some C. lus-itaniae to amphotericin, and certainother species may have acquired resis-tance mechanisms, including C. albicans with resistance to azoles, and the incon-sistent or marginal susceptibility of C. glabrata , C. tropicalis , and C. dublinien- sis to the azoles (277). Acquired azoleresistance has also occurred in Crypto-coccus neoformans in acquired immuno-deciency syndrome patients receivingprolonged azole prophylaxis. A referencebroth microdilution susceptibility testhas been developed and standardized bythe NCCLS for determining the MICs of most antifungal agents with yeast fungi(278). However, those tests require atleast 48 hrs to obtain results and mayonly be available in large reference labo-ratories. More recently, the NCCLS hasdescribed a simple disk diffusion screen-ing test for uconazole resistance in Can-dida species that requires only 24 hrs andis simple to perform by laboratories of allsizes (279)). Basic reference procedureshave also been developed for susceptibil-ity testing of some mould fungi (280),although such methods are performedonly in selected reference laboratories atthis time.

Summary Recommendations

1. All patients with suspected severesepsis should have blood cultures taken.This is true whatever the suspectedsource, although in some cases (e.g., cel-lulitis, see below) the yield is low. Fever,chills, hypothermia, leukocytosis, leftshift of neutrophils, neutropenia, and thedevelopment of otherwise unexplainedorgan dysfunction, e.g., renal failure orsigns of hemodynamic compromise, arespecic indications for obtaining blood

for culture. Blood cultures should betaken as soon as possible after the onsetof fever or chills. In patients with sus-pected catheter-related infection, a pairof blood cultures obtained through thecatheter hub and a peripheral site shouldbe obtained simultaneously. When severesepsis develops in patients colonized byCandida species at two or more sites,blood cultures should be taken for the

detection of candidemia; it is uncertain if the lysis-centrifugation method increasesthe sensitivity for detecting candidemiain this setting.

Grade E

2. Blood should be obtained for cul-ture from a peripheral vein wheneverpossible and a protocol for skin prepara-tion and sampling adopted to minimizethe risk of contamination. The blood cul-ture stopper should also be disinfectedbefore inoculation. Inoculating at least

10 mL of blood per bottle (at least 20 mLtotal per culture) is recommended to op-timize the sensitivity of cultures. If insuf-cient blood is available or if anaerobicinfection is unlikely given the clinicalcontext, only the aerobic bottle should beinoculated.

Grade B3. Blood drawn through catheters is at

higher risk of contamination than whendrawn from a peripheral vein, and thelatter is recommended. Only when pe-ripheral blood is too difcult to obtainshould catheter blood only be cultured,unless catheter-related infection is sus-pected; in the latter case, both peripheralblood and catheter blood should be cul-tured and their time to positivity com-pared.

Grade D

4. Two (maximum three) sets of bloodcultures are sufcient and should be ob-tained at the initial evaluation of eachepisode of suspected bacteremia.

Grade D5. In critically ill septic patients, in

whom it may not be possible to delaytreatment, no interval is required be-tween taking two or three sets of bloodcultures.

Grade D

6. Signs of inammation at the cath-eter entry site are neither sensitive norspecic to suggest infection; most central

venous catheter infections occur in theirabsence, and their presence should notlead to catheter removal. However, whenpresent, a few signs and symptoms arehighly suggestive of catheter infection ina septic patient, including gross puru-lence at the catheter insertion site, cellu-litis extending 4 mm around the site, ortunnel tract infection. Gross purulence atthe CVC site should prompt catheter re-

placement at a new site, irrespective of culture results.

Grade D

7. Cultures of catheter segment andblood are the only means to prove ordisprove denitely catheter-related infec-tion and bacteremia. When catheter in-fection is suspected, swabs should betaken from the insertion site for culture.Such samples have high negative predic-tive value for systemic infection, but theirpositive predictive value is low, especially

when coagulase-negative staphylococciare recovered, unless semiquantitativecultures are obtained. Routine surveil-lance cultures of the catheter insertionsite are not recommended. Hub culturesmay be performed for long-term cathe-ters and those used for parenteral nutri-tion, as the frequency of intraluminal col-onization increases with the catheterindwelling time. Their predictive value iscomparable with that of skin site cul-tures. When bacteremia is suspected andthe catheter can be left in place (no frank

local signs, no severe sepsis), both pe-ripheral and catheter-blood culturesshould be performed and their differen-tial time to positivity measured.

Grade D

8. When a CVC is suspected as a sourceof sepsis in a nonbacteremic patient, de-nitive diagnosis requires that the CVCshould be removed and sent for culture,in selected cases after catheter exchangeover a guidewire. A denite diagnosis of nonbacteremic catheter-related sepsis re-

quires that the catheter proves colonized,that there is no other apparent source of sepsis, and that sepsis abates within 48hrs of catheter removal in the absence of specic antibiotic therapy.

Grade E

9. The strategy adopted to investigatea suspected CRI depends on the clinicalseverity, the presence of bacteremia, andthe organisms involved. In patients withsevere sepsis and suspected CRI, espe-




cially when bacteremia due to one of the“high-risk” organisms ( S. aureus , Can-dida , Pseudomonas ) occurs, the cathetershould be removed without delay. When aCVC is suspected as a source of bactere-mia, diagnosis of CVC infection may bemade by blood culture-based techniques without catheter removal 1) if the pa-tient’s clinical condition permits a poten-tially infected catheter to be left in place,

2) conserving the catheter is a primaryobjective and treatment of CVC infectionin situ is considered, or 3) other potentialsources of bacteremia are apparent. Thisapproach to clinical diagnosis has beenassessed in two ways: 1) by quantitativeor semiquantitative paired blood cul-tures; 2) by measuring time to culturepositivity in continuous monitoringblood culture systems. Being culture-based methods, both involve a delay of 24– 48 hrs before cultures results areavailable, a time interval when a poten-tially infected catheter remains in situ .Their applicability to the context of thecritically ill ICU patients has not been well assessed. Guidewire exchange of thecatheter can be performed in selectedcases.

Grade D

10. Blood cultures and respiratory se-cretions (sputum or endotracheal aspi-rates) must be obtained. Urinary antigendetection of Legionella serogroup 1 isrecommended in endemic areas or dur-ing outbreaks. Pleural uid for Gram-negative stain and culture should be ob-tained if available. Serology may provideevidence of a microbial etiology in somecases. Invasive procedures are optionaland should be restricted to intubated pa-tients. A diagnostic algorithm is pro-posed.

Grade D

11. Up to 10% of patients with CAP donot respond to initial antibiotic therapy.Mortality in this population is very high(40% to 50%). A complete microbiologi-cal reevaluation is recommended (Table7). Invasive techniques including bron-choscopy with a protected specimenbrush and bronchoalveolar lavage shouldbe performed. Chest CT scans may beuseful in some patients.

Grade D

12. VAP is dened as pulmonary inl-trates plus two of the following: fever

38°C, leukopenia, leukocytosis, or pu-rulent secretions, or a CPIS of 6.

The presence of new chest radiographinltrates plus two of the three clinical variables (fever 38°C, leukocytosis orleukopenia, and purulent secretions) isuseful for the clinical screening of VAP(high sensitivity). The modied clinicalpulmonary infection score ( 6) is alsouseful for initial screening.

However, for patients suffering fromacute respiratory distress syndrome andfor whom it is difcult to demonstratedeterioration of radiologic images, atleast one of the three preceding clinicalcriteria may sufce to activate initialscreening. A high index of suspicionshould also be maintained in cases of unexplained hemodynamic instability ordeterioration of blood gases in patientsrequiring mechanical ventilation.

The combination of the presence of pulmonary inltrates plus two of the

three clinical signs is sufcient to con-sider initiating antibiotic treatment. A modied CPIS of 6 can be also used forthis purpose. In both cases, additionalprocedures, such as cultures of lower re-spiratory tract secretions, are needed be-fore starting therapy.

Grade D

13. The preferred specimens for eval-uation of lower tract secretion are quan-titative or semiquantitative cultures of endotracheal aspirates, blind or broncho-

scopic PSB, or BAL samples. The detec-tion of intracellular organisms ( 5%) inBAL uid is a very specic marker for VAP.

Quantitative or semiquantitative cul-tures of nonbronchoscopic (endotrachealaspirates or blind PSB or BAL) or bron-choscopic techniques are preferred(bronchoscopic PSB and BAL). Becausethese tests have similar sensitivities,specicities, predictive values, and likeli-hood ratios, the choice depends on localexpertise, experience, availability, andcost factors. The nding of 5% of intra-cellular microorganisms in centrifugedBAL uid is a highly specic marker of VAP.

Grade B

14. A quantitative strategy for the di-agnosis of VAP is preferred.

Grade B

A clinical strategy is appropriate whenquantitative cultures are not available,

although this is less specic than thequantitative strategy.

Grade B

In both cases, samples should be ob-tained before new antibiotic administra-tion.

Grade D

A negative culture of an endotracheal

aspirate sample without prior antibiotictreatment rules out VAP.

Grade D

15. In patients with clinical evidenceof severe sepsis or patients with a veryhigh pretest probability of the disease,the initiation of antibiotic therapy shouldnot be delayed and patients should betreated immediately with broad-spectrumantibiotics, even when no bacteria aredetected using microscopic examinationof pulmonary secretions.

Grade E

16. When quantitative culture tech-niques are not available in patients clin-ically suspected of having HAP, we rec-ommend following a clinical strategy,emphasizing that antimicrobial treat-ment should be re-evaluated on day 3, when susceptibility patterns of the micro-organism(s) considered to be causativeare available, to select treatment with anarrower spectrum or even to stop anti-microbial therapy if the clinical picture is

barely compatible with a diagnosis of pneumonia. In both strategies, samples of respiratory secretions for culturingshould be obtained before antibiotic ad-ministration or antibiotic changes forpneumonia to avoid false-negative re-sults. In addition, a negative culture of respiratory secretions in the absence of prior antibiotic treatment rules out bac-terial pneumonia in most cases.

Grade E

17. In general, when draining a soft-

tissue infection, the rst approach is toperform a Gram stain and bacterial cul-ture of any obvious wound exudate or toperform a needle aspiration of a closedinfection, e.g., cellulitis or abscess forstain and bacterial culture.

Grade E

18. Ultrasonography or magnetic res-onance imaging may be helpful in thediagnosis of some cases of cellulitis or toguide needle aspiration of the site but




should not be allowed to delay the diag-nosis by needle aspiration or delay sur-gery needed to debride infected tissue.

Grade E

19. Patients with severe skin and softtissue infection should have blood cul-tures obtained, although these are onlyrarely useful in the diagnosis of mild(nonnecrotizing) cellulitis.

Grade E

20. In most situations ultrasound willbe the rst modality of choice in evalua-tion of skin infections. When ultrasoundis not diagnostic, CT scanning should beperformed.

Grade E

21. Collections identied by imagingstudies should, if possible, be aspiratedand drained during imaging and a sub-stantial volume of uid collected for rapid

Gram staining and for both aerobic andanaerobic cultures.

Grade E

22. Abdominal drain specimens may yield misleading results due to supercialcontamination of the distal portions of drains. Excess antibiotic use could wellresult from the reliance on culture datafrom surgical drains.

Grade E

23. Blood cultures are recommended

routinely with intraabdominal infections(two sets of blood cultures should be ob-tained).

Grade E

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