254

REV. HOSP. CLÍN. FAC. MED. S. PAULO 58(5):254-259, 2003

From the Division of Respiratory Diseases,Heart Institute (INCOR), Hospital dasClínicas, Faculty of Medicine, University ofSão Paulo – São Paulo/SP, Brazil.

Received for publication onMarch 17, 2003.

PREVALENCE RATES OF INFECTION IN INTENSIVECARE UNITS OF A TERTIARY TEACHING HOSPITAL

Carlos Toufen Junior, André Luiz Dresler Hovnanian, Suelene Aires Franca andCarlos Roberto Ribeiro Carvalho

TOUFEN Jr. C et al. - Prevalence rates of infection in intensive care units of a tertiary teaching hospital. Rev. Hosp. Clín. Fac.Med. S. Paulo 58 (5):254-259, 2003.

OBJECTIVE: To determine the prevalence rates of infections among intensive care unit patients, the predominantinfecting organisms, and their resistance patterns. To identify the related factors for intensive care unit-acquired infectionand mortality rates.

DESIGN: A 1-day point-prevalence study.SETTING: A total of 19 intensive care units at the Hospital das Clínicas - University of São Paulo, School of Medicine

(HC-FMUSP), a teaching and tertiary hospital, were eligible to participate in the study.PATIENTS: All patients over 16 years old occupying an intensive care unit bed over a 24-hour period. The 19

intensive care unit s provided 126 patient case reports.MAIN OUTCOME MEASURES: Rates of infection, antimicrobial use, microbiological isolates resistance patterns,

potential related factors for intensive care unit-acquired infection, and death rates.RESULTS: A total of 126 patients were studied. Eighty-seven patients (69%) received antimicrobials on the day of

study, 72 (57%) for treatment, and 15 (12%) for prophylaxis. Community-acquired infection occurred in 15 patients (20.8%),non- intensive care unit nosocomial infection in 24 (33.3%), and intensive care unit-acquired infection in 22 patients(30.6%). Eleven patients (15.3%) had no defined type. The most frequently reported infections were respiratory (58.5%).The most frequently isolated bacteria were Enterobacteriaceae (33.8%), Pseudomonas aeruginosa (26.4%), andStaphylococcus aureus (16.9%; [100% resistant to methicillin]). Multivariate regression analysis revealed 3 risk factors forintensive care unit-acquired infection: age ≥ 60 years (p = 0.007), use of a nasogastric tube (p = 0.017), and postoperativestatus (p = 0.017). At the end of 4 weeks, overall mortality was 28.8%. Patients with infection had a mortality rate of 34.7%.There was no difference between mortality rates for infected and noninfected patients (p=0.088).

CONCLUSION: The rate of nosocomial infection is high in intensive care unit patients, especially for respiratoryinfections. The predominant bacteria were Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus(resistant organisms). Factors such as nasogastric intubation, postoperative status, and age ³60 years were significantlyassociated with infection. This study documents the clinical impression that prevalence rates of intensive care unit-acquiredinfections are high and suggests that preventive measures are important for reducing the occurrence of infection in criticallyill patients.

DESCRIPTORS: Intensive care unit. Nosocomial infection. Prevalence rate.

Intensive care units (ICUs) are ofparamount importance in the controland treatment of the most variable andsevere illnesses that affect the humanbody. They represent a powerful toolin modern medicine.

In the beginning, beds for intensivecare were established to provide post-operative care for critical patients in

1920s. During the Second World War,they became more prevalent. The firstICUs were created in the 1950s to treat

patients who needed special monitor-ing during the postoperative period ofcardiac surgery. In 1971, the Societyof Critical Care Medicine wasfounded, and during this decade, inten-sive care medicine reached the posi-tion of a medical specialty.

In spite of their invaluable andwell-established role, ICUs bring some

255

REV. HOSP. CLÍN. FAC. MED. S. PAULO 58(5):254-259, 2003 Prevalence rates of infection in intensive care unitsToufen Jr. C et al.

degree of morbidity to patients, andnosocomial infection is clearly relatedto that.

Nosocomial infection is associatedwith a considerable increase in morbid-ity and mortality of patients at a hos-pital as well as to significant increasesin costs1. Nosocomial infections occurin 5% to 17% of hospitalized pa-tients1. In ICUs, where the frequent useof invasive procedures and multipletherapies expose patients to an in-creased risk, prevalence rates are evenhigher1.

Data from the Study on the Effi-cacy of Nosocomial Infection Control(SENIC) show that one-third of noso-comial infections could be preventedthrough infection control and vigi-lance programs2.

In view of the relevance and impactof such observations, it is crucial toknow the prevalence rates and natureof nosocomial infections to achievesatisfactory results in controlling thisimportant phenomenon.

The present study was undertakento determine the prevalence rates ofinfection for ICU patients in our hos-pital, identify the most common infec-tious agents and their resistance pat-terns, and establish the prevalence ratesof ICU-acquired infection and the pos-sible associated factors.

PATIENTS AND METHODS

Setting. A total of 19 ICUs at ateaching and tertiary hospital were eli-gible to participate in the study.Pediatric intensive care and infant spe-cial care units were excluded from thestudy. The study period lasted 24hours during June 15, 2000. Seventeenvolunteers (medical doctors and stu-dents of medicine) participated in theexperiment collecting data on a surveyrecord form.

Patients. All patients over 16 yearsof age occupying a bed in a participat-

ing ICU over the study period tookpart in the study. For each patient, in-formation was collected concerningdemographics (age and gender), opera-tive and clinical status on admissionto the ICU, and diagnostic and thera-peutic interventions performed on thestudy day. The Simplified AcutePhysiologic Score II (SAPS II)3 was cal-culated from data collected in the first24 hours of ICU admission.

Types of infection. The presence orabsence of infection by type was docu-mented according to the standard defi-nitions of the Centers for Disease Con-trol and Prevention (CDC):• Community-acquired – an infec-

tion occurring in the communityand manifested on admission tohospital;

• Hospital-acquired – an infectionmanifested on admission to theICU and deemed to be related tothe present hospital admission;

• ICU-acquired – an infection hav-ing originated in the ICU and ac-tive or under treatment on the dayof study but not clinically mani-fested at the time of admission tothe ICU.Antimicrobials prescribed on June

15, whether for treatment or prophy-laxis, were documented. Microbiologi-cal data were recorded whenever avail-able, including the results of bacterio-logic sampling undertaken on or be-fore the day of study.

Recorded risk factors for ICU-ac-quired infection included the presenceor absence of intravascular and urinarycatheters, nasogastric intubation, me-chanical ventilation, and agents forstress ulcer prophylaxis.

Patient outcome was recorded byresearching patient data 4 weeks afterthe study day.

Following the completion of thestudy, all record forms were collectedcentrally. Data entry was followed bya series of computer validation tests todetect omissions or inconsistent en-

tries. Any errors identified at this stagewere corrected whenever possible orthe data were recorded as “missing”.

Variables. Some potential risk fac-tors for ICU-acquired infection wereanalyzed. These included: age equalto or greater than 60 years; kind of ad-mission (medical or surgical); time pe-riod of ICU stay; and use of a centralvenous catheter, nasogastric tube, uri-nary catheter, and/or gastric protectors.

Definition of infection. A patientwas given a diagnosis of infectionwhen there was a medical diagnosis ofan infectious focus that required theuse of antimicrobial treatment.

Cultures. The cultures employedwere: cultures of blood (more than 2positive pairs of culture for the samepathogen), urine (more than 10,000CFU when collected from urinary cath-eter and 100,000 CFU when not),broncho-alveolar lavage (more than10,000 CFU for a single pathogen),and surgical wounds.

Statistical analysis. The pointprevalence of community-acquired,hospital-acquired, and ICU-acquiredinfections was estimated. Data from theoverall population were used foranalysis of infection. The risk factorsfor ICU-acquired infection were esti-mated at first with a simple unadjustedanalysis. Aiming at adjusting the vari-ables and controlling the effect of po-tentially confounding variables, amultivariate model construction by lo-gistic regression analysis was per-formed. Epi Info, version 6.04, andSPSS, version 6.0.1, were used.

RESULTS

ICU Profiles. Among the 19 ICUs,with 170 beds, 1 did not participate inthe study because there were no pa-tients on the day of study. In the re-maining 18, there were 126 patients.

Patients’ Demographics and Inter-ventions. Among the 126 patients

256

REV. HOSP. CLÍN. FAC. MED. S. PAULO 58(5):254-259, 2003Prevalence rates of infection in intensive care unitsToufen Jr. C et al.

studied, 70 were male (56%); the av-erage age was 56.6 + 18.1 (SD) years.Female patients accounted for 44% ofall patients; the average age was 59.2+ 17.2 (SD) years.

A total of 52 patients had under-gone surgery 24 hours before admis-sion, of whom 38 (73%) had electiveand 14 (27%) had emergency surgery.

On the day of the study, 52 patients(41.3%) were being mechanically ven-tilated, 86 (68.2%) had a central ve-nous catheter, 10 (7.9%) had a Swan-Ganz catheter, 88 (69.8%) had a uri-nary catheter, and 66 (52.3%) had anasogastric tube (Figure 1).

Antimicrobial Use. A total of 87patients (69%) received antimicrobialson the day of the study, 72 (82.8%) fortreatment and 15 (17.2%) for prophy-laxis. The most frequently used antimi-crobials were cephalosporins (48.6%),followed by vancomycin (45.8%) andcarbapenems (25%).

Prevalence of infection. Among the72 patients who received therapeuticantibiotics, 15 (20.8%) had commu-nity-acquired infection, 24 (33.3%)had acquired an infection at anotherhospital service before being trans-ferred to the ICU, 22 (30.6%) had ICU-acquired infection, and 11 (15.3%)had no defined type (Figure 2).

Regarding the site of infection, 41(56.9%) were pulmonary, 5 (7.0%)were clinical sepsis, and 4 (5.6%) hadan infection site in the urinary tract(Figure 3).

Overall, 37 (51.4%) of the clini-cally identified infections were sup-ported by positive cultures. Fourteenpatients had a mismatch between theantimicrobial used and the antibio-gram, representing 19.4% of the 72 pa-tients that were considered to be clini-cally infected. This inadequate antimi-crobial treatment of infection was ob-served more frequently among patientswith ICU-acquired infection (22.7%),followed by patients with community-acquired infection (20.0%), and ac-

quired infection at another hospitalservice before being transferred to ICU(16.6%).

The most frequently reported iso-lates were as follows: Enterobacte-riaceae (33.8%), Pseudomonasaeruginosa (26.4%), Staphylococcus

aureus (16.9%), Streptococci (7.5%),coagulase-negative staphylococci(5.6%), and Candida species (7.5%).

Patterns of Antimicrobial Resist-ance. Among the 9 S. aureus isolates,all were methicillin-resistant strains(MRSAs), and all were sensitive to

UC- Urinary Catheter; CVC- Central Venous Catheter; NGT- Nasogastric Tube; MV-

Mechanical Ventilation; SGC- Swan-Ganz Catheter.

Figure 1 - Invasive procedures of 126 patients studied on June 15, 2000.

Figure 2 - Prevalence rates of infection by type of 72 patients who received therapeuticantibiotics on June 15, 2000.

257

REV. HOSP. CLÍN. FAC. MED. S. PAULO 58(5):254-259, 2003 Prevalence rates of infection in intensive care unitsToufen Jr. C et al.

vancomycin. Among the 14 culturespositive for P. aeruginosa, 12 weretested for gentamicin, with 50% resist-ance; 10 for imipenem, with 30% re-sistance; 11 for ceftazidime, with 50%resistance; 12 for ciprofloxacin, with41.6% resistance; and 12 for amikacin,with 41.6% resistance.

Risk Factors for ICU-Acquired In-fection. Univariate analysis was firstemployed to identify possible risk fac-tors for acquiring infection inside theICU environment. Only age equal to orgreater than 60 years was identified asa risk factor. After stepwise logistic re-gression analysis to control for the ef-fects of confounding variables, 3 fac-tors were independent risk factors: useof a nasogastric tube (p = 0.017), post-operative status (p = 0.017,) and ageequal to or greater than 60 years (p =0.007) (Table 1).

Mortality. At the end of 4 weeks,overall mortality was 28.8%. Patientswith infection had a mortality rate of34.7%. Using a chi-square analysis, wefound no difference between mortality

rates for infected and noninfected pa-tients (p = 0.088).

DISCUSSION

The present study, through a trans-versal (1-day point prevalence) analy-sis, reveals a profile of patient infec-tions in the ICUs of a tertiary teach-ing hospital. It aims at characterizingpatients with a focus on the prevalencerates of infection, sites and types of in-fection, the most prevalent microorgan-

isms, and the antimicrobial resistancepatterns, as well as at identifying therisk factors for ICU-acquired infection.

Medical staff that assisted the pa-tients in the ICUs found 72 (57.1%)infected; thus, they prescribed antimi-crobials with therapeutic intention.Similar results were obtained in aMexican study (58.2%)5. The EPIC,a European study, showed lower ratesof infection (44.8%)1. The great major-ity of patients (73.8%) received anti-microbials. This finding shows thatbecause of the difficulty in establish-ing the specific diagnosis and becauseof the severity of infection, antimicro-bials are empirically introduced mostof the time. Among this population, 15(20.8%) had community-acquired in-fection, 24 (33.3%) had infections thatwere hospital-acquired outside theICU, and 22 (30.6%) developed infec-tion inside the ICU. The remainder 11infected patients (15.3%) were not clas-sified. Higher prevalence rates for ICU-acquired infections are reported in theliterature1,5.

Overall, 37 (51.4%) of the clini-cally identified infections were sup-ported by positive cultures. These iso-lates might not necessarily have rep-resented the cause of the infection.They might have only reflected possi-ble contamination of the sample or theprocess of colonization. Nevertheless,regarding the isolates reported, thepredominance of gram-positive cocci,particularly S. aureus, and gram-nega-

Table 1 - Risk factors for Intensive Care Unit-acquired infection after logisticregression analysis.

Variable p value

Medical admission 0.78Surgical admission† 0.017Time period of Intensive Care Unit stay 0.79Use of Central Venous Catheter 0.58Use of Nasogastric Tube† 0.017Use of Urinary Catheter 0.76Use of gastric protectors 0.72Age ≥ 60 years† 0.007SAPS II 0.87

† Independent risk factors.

Figure 3 - Prevalence rates of infection by site of 72 patients who received therapeutic antibioticson June 15, 2000.

258

REV. HOSP. CLÍN. FAC. MED. S. PAULO 58(5):254-259, 2003Prevalence rates of infection in intensive care unitsToufen Jr. C et al.

tive organisms, especially P.aeruginosa, are in accordance withfindings of recent studies1,5. Fourteen(11.1%) of the patients received inad-equate treatment on the day of thestudy. This represents 19.4% of the 72patients that were considered to beclinically infected. This finding couldbe explained by 2 possibilities: mis-taken diagnosis by the medical teamor failure of the diagnostic method ifthe medical diagnosis was correct.

Inadequate antimicrobial treatmentof infection was observed more fre-quently among patients with ICU-ac-quired infection (22.7%), followed bypatients with community-acquired in-fection (20.0%), and acquired infec-tion at another hospital service beforetransfer to the ICU (16.6%).

It is interesting that 56.9% of pa-tients were being treated for pneumo-nia, a number considerably higher thanfor other infections. This finding re-veals the concern of medical teamswith pneumonia, maybe because of itshigh morbidity and mortality rates andits known high prevalence rates. Ad-ditionally, it is quite difficult to dis-tinguish nosocomial pneumonia frombacterial tracheobronchial coloniza-tion using only clinical findings, es-pecially in intubated patients6. Defini-tive diagnosis that pneumonia is theresult of a specific pathogen can beobtained only if cultures of blood,pleural fluid, or spinal fluid are posi-tive in the presence of a lung infiltrateand a compatible clinical picture7.Bacteremia is uncommon in mostpneumonias, usually occurring in only8% to 15% of nosocomial pneumo-nias8,9.

Regarding bacterial agents, themost prevalent were a member of thefamily of Enterobacteriaceae, Pseu-domonas aeruginosa, andStaphyloccocus aureus, which were re-spectively 33.8%, 26.4%, and 16.9%of the isolated agents. Concerning re-sistance patterns for S. aureus, 100%

were resistant to methicillin and 100%had sensitivity to vancomycin. Amongcultures of P. aeruginosa, none re-vealed sensitivity higher than 70% toany antimicrobial tested. These dataconfirm that in the intensive caremedical environment, the most patho-genic agents, those that are in generalthe most resistant, are the most fre-quently found1,5. Regarding use ofantimicrobials, the most frequentlyused were cephalosporins (35 patients,48.6%), vancomycin (33, 45.8%),imipenem (18, 25%), clindamycin (8,11.1%), and aminoglycosides (6, 8.3%).Normally, empirical treatment schemesare based on knowledge of localmicrobiota and utilization of a widerange of antimicrobials associated ornot with others of more specific ac-tion, such as vancomycin for S. aureusor clindamycin for anaerobic bacteria.

Three factors were significantly re-lated to ICU-acquired infection: use ofa nasogastric tube, postoperative sta-tus, and age equal to or greater than60 years.

The finding of a relationship be-tween nasogastric intubation and in-fection is in accordance with the lit-erature7,10. According to the literature,pneumonia was the most frequentlyobserved infection in ICUs, and naso-gastric intubation represents one ofthe main risk factors. Aspiration oforopharyngeal contents, which is richin bacteria, is facilitated by the pres-ence of a nasogastric tube, particularlyif food is administered in bolus orwhen the patient is in a supine posi-tion. Use of drugs that increase gastricpH, thus facilitating growth of gram-negative bacteria, raises the risk of pul-monary infections. Our data show thatthese drugs were used frequently (107patients – 85.6% – were given gastricprotectors, while only 6 patients witha nasogastric tube were not), in ac-cordance with the relationship pointedout in the literature7.

Postoperative patients also had a

higher number of infections. This typeof patient has a higher propensity fordeveloping infectious complications,since surgical incision represents agateway for infection. Additionally,postoperative patients are intubatedfor surgery and have a transitory de-crease in immune responses due to sur-gical damage.

Patients of an age equal to orgreater than 60 years have higher ratesof ICU-acquired infections, a findingthat reveals the fragility of the elderlyto infections and invasive procedures.

Our data reveal frequent use ofantimicrobials in our intensive caremedicine environment, even withoutlaboratory support for it. This findinghas potential implications, in thattreatment with antimicrobials raisesthe cost of treatment considerably andcontributes to the selection of multire-sistant species. The excessive use ofgastric protectors was unexpected,since there is no evidence that their usewould bring any benefit in terms of re-duction of digestive complications.Simple practices could be immediatelyperformed at ICUs to reduce rates ofpulmonary infections, such as keepingthe head of the bed elevated and tak-ing care with diet administration.

It should be noted that a preva-lence study like this one has somelimitations. One limitation concernsthe difficulty in establishing relation-ships of causality between factors; onecannot predict which factor comesfirst. The other is that 1-day point stud-ies tend to overestimate long-durationinfections and underestimate the short-duration ones.

Nevertheless, this type of transver-sal analysis has fundamental impor-tance for the knowledge about theICU population. It provides an impe-tus for new strategies and interven-tions concerning critical patients interms of scientific research and clini-cal practice.

259

REV. HOSP. CLÍN. FAC. MED. S. PAULO 58(5):254-259, 2003 Prevalence rates of infection in intensive care unitsToufen Jr. C et al.

RESUMO

TOUFEN Jr. C e col. - Prevalência deinfecção em unidades de terapiaintensiva de um hospital escolaterciário. Rev. Hosp. Clín. Fac.Med. S. Paulo 58 (5):254-259,2003.

OBJETIVO: Determinar a preva-lência de infecções em pacientes deTerapia Intensiva, os agentes infecci-osos mais comuns e seus padrões deresistência. Identificar os fatores rela-cionados a infecção adquirida na Uni-dade de Terapia Intensiva e as taxas demortalidade.

DESENHO: Estudo de prevalênciade um dia.

LOCAL: Um total de 19 Unidadesde Terapia Intensiva do Hospital dasClínicas da FMUSP (HC-FMUSP) par-ticiparam do estudo.

PACIENTES: Todos os pacientescom idade superior a 16 anos interna-dos em leitos de terapia intensiva pormais de 24 horas foram incluídos. As19 Unidades de Terapia Intensiva for-neceram 126 casos.

VARIÁVEIS: Taxas de infecção,uso de antibióticos, padrões de resis-

tência microbiológica, fatores relacio-nados à infecção adquirida na Unida-de de Terapia Intensiva, taxas de mor-talidade.

RESULTADOS: Um total de 126pacientes foi estudado. Oitenta e sete(69%) receberam antibióticos no diado estudo, sendo 72 (57%) para trata-mento e 15 (12%) para profilaxia. Ba-seado no tipo, observou-se que a in-fecção adquirida na comunidade ocor-reu em 15 pacientes (20,8%), infecçãohospitalar fora da Unidade de TerapiaIntensiva em 24 (33,3%), e infecçãoadquirida na Unidade de Terapia In-tensiva em 22 pacientes (30,6%). Para11 pacientes (15,3%) não se definiu otipo de infecção. Quanto ao sítio deinfecção, as respiratórias foram as in-fecções mais comuns (58,5%). Os agen-tes mais freqüentemente isolados fo-ram: Enterobacteriaceae (33,8%),Pseudomonas aeruginosa (26,4%) eStaphylococcus aureus (16,9%; 100%meticilina-resistentes). Análise multi-variada identificou 3 fatores associa-dos à infecção adquirida na Unidadede Terapia Intensiva: idade maior ouigual a 60 anos (p=0,007), uso de son-

da nasogástrica (p=0,017) e pós-ope-ratório (p=0,017). Ao final de quatrosemanas, a taxa de mortalidade foi de28,8%. Entre os infectados, a mortali-dade foi de 34,7%. Não houve diferen-ça entre as taxas de mortalidade parapacientes infectados e não-infectados(p=0,088).

CONCLUSÃO: A taxa de infecçãoé alta entre os pacientes de terapia in-tensiva, especialmente as infecçõesrespiratórias. As bactérias predominan-tes foram: Enterobacteriaceae. Pseu-domonas aeruginosa e Staphylo-coccus aureus (agentes resistentes).Fatores como uso de sonda nasogás-trica, pós-operatório e idade maior ouigual a 60 anos mostraram associaçãocom infecção. Este estudo documentaa impressão clínica de que a preva-lência de infecção adquirida na Uni-dade de Terapia Intensiva é alta e su-gere que medidas preventivas são im-portantes para reduzir a ocorrênciade infecção em pacientes críticos.

DESCRITORES: Unidade de tera-pia intensiva. Infecção nosocomial.Taxa de prevalência.

REFERENCES

1. VINCENT J-L, BIHARI DJ, SUTER PM et al. - The prevalence ofnosocomial infection in intensive care units in Europe – resultsof the European Prevalence of Infection in Intensive Care(EPIC) Study. JAMA 1995; 274(8): p. 639-44.

2. HALEY RW, CULVER DH, WHITE JW et al. - The efficacy ofinfection surveillance and control programs in preventingnosocomial infections in US hospitals. Am J Epidemiol 1985;121: p. 182-205.

3. LE GALL J-R, LOIRAT P, ALPEROVITCH A et al. - A simplifiedacute physiology score for ICU patients. Crit Care Med 1984;12: p. 975.

4. GARNER J, JARWIS W, EMORI T et al. - CDC Definitions fornosocomial infections. Am J Infect Control 1988; 16: p.128-40.

5. LEÓN-ROSALES SP, MOLINAR-RAMOS F, DOMÍNGUEZ-CHERIT F et al. - Prevalence of infections in intensive careunits in Mexico: a multicenter study. Crit Care Med 2000;28(5): p. 1316-21.

6. SALATA RA, LEDERMAN MM, SHLAES DM et al. - Diagnosisof nosocomial pneumonia in intubated, intensive care unitpatients. Am Rev Resp Dis 1987; 135(2): p. 426-32.

7. NIEDERMAN MS - Acute infectious pneumonia. In: IRWIN RS,CERRA FB, IPPE JM - Intensive Care Medicine. 4th ed. Riode Janeiro, Cidade Editora, 1999. v. 1, p. 879-99.

8. BRYAN CS, REYNOLDS KL - Bacteremic nosocomialpneumonia: analysis of 172 episodes from a single metropolitanarea. Am Rev Resp Dis 1984; 129: p. 668.

9. BATES JH, CAMPBELL GD, BARRON AL et al. - Microbialetiology of acute pneumonia in hospitalized patients. Chest1992; 101: p. 1005.

10. LEAL-NOVAL SR, MARQUEZ-VACARO JA, GARCIA-CURIELA et al. - Nosocomial infection in patients undergoing heartsurgery. Crit Care Med 2000; 28(4): p. 935-400.