Binelli Et Al. 2006 Infecções Urinárias

ORIGINAL ARTICLE 10.1111/j.1469-0691.2006.01435.x

Investigation of the possible association between nosocomial candiduriaand candidaemiaC. A. Binelli1, M. L. Moretti2, R. S. Assis1, N. Sauaia1†, P. R. Menezes1, E. Ribeiro2, D. C. P. Geiger2,Y. Mikami3, M. Miyaji3, M. S. Oliveira2, A. A. Barone1 and A. S. Levin1

1Faculty of Medicine of the University of Sao Paulo, Sao Paulo, 2Faculty of Medical Sciences, StateUniversity of Campinas, Campinas, Brazil, and 3Chiba University, Chiba, Japan

ABSTRACT

This study aimed to determine whether candiduria is associated with the occurrence of nosocomialcandidaemia. In the case-control part of the study, 115 cases (nosocomial candidaemia) and 115 controls(nosocomial bacteraemia) were similar in age, severity of condition and time of hospitalisation. Therewas a significant association of candidaemia with candiduria (OR 9.79; 95% CI 2.14–44.76). In themicrobiology part of the study, 23 pairs of Candida-positive urine and blood cultures were obtained from23 patients. In ten (43%) cases, the urine and blood culture isolates belonged to different species, andmolecular typing showed a difference in two of the 13 cases yielding the same species from bothspecimens. Overall, there was a significant association between candiduria and candidaemia, but theCandida isolates from urine and blood were different for 52% of the patients. Thus, the data indicatedthat the urinary tract was probably not a source for the candidaemia.

Keywords Bacteraemia, Candida spp., candidaemia, candidurea, nosocomial infection

Original Submission: 9 June 2005; Revised Submission: 22 September 2005; Accepted: 10 November 2005

Clin Microbiol Infect 2006; 12: 538–543

INTRODUCTION

Candida spp. frequently colonise the digestive andgenital tracts, as well as the mucosal surfaces, of2–37% of healthy individuals, but colonisation of80% of hospitalised patients can occur [1,2].Colonisation usually precedes candidaemia, andis considered to be an important risk-factor asmost infections are endogenous [1–7]. Candidae-mia is a severe infection and has a high risk ofdeath (46–80%) when compared with bacteraemia[1,8–10], with an attributable mortality of 38% [8].

The natural history of candiduria is not clearlyestablished. Urinary tract colonisation deservesconsideration, as it is a common event in hospi-talised patients [11–14], affecting 6.5–20% ofpatients [5]. Candiduria, which is asymptomaticor with only slight symptoms, is consideredbenign, even in patients with underlying disease

such as diabetes mellitus [15,16]. Patients withurological disease and candiduria who undergosurgery or manipulation of the urinary tract are atrisk of candidaemia [17], considered to be ascend-ant and not haematogenic [18]. However, somestudies emphasise that candiduria should neverbe ignored in septic patients, as it may be the firstsign of a systemic Candida infection [19–21].However, it seems to be difficult to differentiatebetween colonisation and infection, and to estab-lish the role of Candida colonisation in the occur-rence of candidaemia. In a multicentre study with4276 intensive care patients [22], rectal and urin-ary colonisation had a low positive predictivevalue for candidaemia, with only 1% of colonisedpatients developing Candida bloodstream infec-tions. Molecular typing demonstrated that thecolonising isolate and that from the blood wereidentical for 17 of 18 Candida infections. In aprospective multicentre study of 861 patients withdocumented funguria, only 1.3% presented withproven candidaemia [16]. The aim of the presentstudy was to determine whether there is anindependent association between candiduria andcandidaemia.

Corresponding author and reprint requests: A. S. Levin, RuaHarmonia 564 ⁄ 52, Sao Paulo, SP 05435–000, BrazilE-mail: [email protected]†Deceased

� 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

METHODS AND MATERIALS

This study comprised two parts: an epidemiological case-control study performed between 1996 and 2000 to evaluatethe association between candiduria and candidaemia, and amicrobiological study involving specimens obtained between1999 and 2001 to evaluate whether Candida spp. isolated fromthe blood and urine of each patient were identical.

Epidemiological study design

Individually matched cases and controls were patients from theHospital das Clınicas of the University of Sao Paulo, Brazil, atertiary-care teaching hospital associated with the universitywith c.2000 beds in five buildings and 15 intensive care units.This institution is the main reference hospital in Sao Paulo, a citywith c.11 million inhabitants. Cases and controls were obtainedfrom the database of the hospital’s infection control department.

A case was defined as a patient aged ‡ 5 years, with at leastone blood culture positive for Candida spp., a Candida-positiveurine culture collected in the period between 2 weeks beforeand 2 days after the positive blood culture was taken, and whowas hospitalised for ‡ 48 h before the positive blood culturewas taken.

A control was defined as a patient aged ‡ 5 years from thesame unit of the hospital as the case, who had a positive bloodculture for bacteria within 9 months of the case, a positiveurine culture collected in the period between 2 weeks beforeand 2 days after the positive blood culture was taken, and whowas hospitalised for ‡ 48 h before the positive blood culturewas taken. When there were several potential controls for acase, the age and date of hospitalisation were selected to be asclose as possible to those of the case.

Assessment of disease and exposure. The following data werecollected from the medical records for cases and controls:gender, age, hospital unit, days of hospitalisation before thepositive blood culture, use of an endotracheal tube during theprevious 30 days, use of central venous and urinary cathetersduring the previous 30 days, use of antimicrobial drugs duringhospitalisation, use of steroids (defined as a daily dose of‡ 15 mg of prednisone or equivalent for ‡ 1 week), chemother-apy or antifungal drugs, dialysis, parenteral nutrition withinthe 30-day period before the positive blood culture, cancer,surgical procedures, care at a day-hospital within 30 days ofthe positive culture, the presence of neutropenia (defined as aneutrophil count < 1000 ⁄mm3) within the previous 2 weeks,and the severity of each patient’s condition on admission andon the day of the positive blood culture according to theAPACHE II score [23]. The isolation of fungi at other sites(defined as any positive cultures other than from blood) wasalso recorded. Underlying diseases were those reported by theattending physician and stated clearly in the patient records.

Candidaemia was defined as a blood culture positive forCandida; candiduria was defined as a urine culture positive forCandida with ‡ 10 000 CFU ⁄mL [24].

Statistical analysis. The sample size was calculated consideringone control for each case, using Stata 7.0 software (Stata Corp.,College Station, TX, USA). The OR to be detected was 3.5 and,based on data from the same hospital in 1997, the prevalence ofcandiduria in patients with bacteraemia was predicted to be

7%. A sample of 115 cases and 115 controls would then have apower of 82% to detect an OR of ‡ 3.5 with p 0.05. A databasewas constructed using Epi Info 6 v.6.02 (CDC, Atlanta, GA,USA). In the univariate analysis, ORs for individually matchedcase-control studies and 95% CIs were estimated for each ofthe categorical variables, and were analysed using McNemarchi-square tests. For continuous variables, the mean, medianand standard deviation were calculated for cases and controls,and the Wilcoxon matched pairs signed-rank test was used foranalysis. Significance was set at p 0.05.

In the multivariate analysis, conditional multiple logisticregression was used. Exposure variables were tested if pwas £ 0.25 in the univariate analysis, using a stepwiseforward method. To stay in the final model, exposurevariables required p values £ 0.05 according to the likelihoodratio test.

Microbiological study

Between 1999 and 2001, all urine cultures positive for Candidaspp. from patients aged ‡ 5 years were stored for 3 weeks. If,within this period, there was a positive blood culture forCandida spp., a matched pair was formed. Isolates from bloodand urine were identified by colony morphology, cell mor-phology, the germ tube test [25], the automated Vitek system(bioMerieux, Marcy l’Etoile, France), CHROMagar [26], carbonassimilation tests (ID32 C; bioMerieux) [27,28], and specificantisera for Candida (Candida check; Iatron, Tokyo, Japan) [29].Molecular typing was performed when the same species ofCandida was isolated from the blood and the urine of a singlepatient.

DNA extraction for randomly amplified polymorphic DNA(RAPD) typing was performed as described previously [30,31],with minor modifications. Four primers were used: Oligo 2primer (5¢-TCACGATGCA) for Candida albicans, Candida tropi-calis and Candida glabrata; Oligo 3 primer (5¢-ATCGATCSSC)for C. albicans and C. glabrata; P5 primer (5¢-AACGCGCAAC)for C. tropicalis; and Oligo 1 primer (5¢-ATTGCGTCCA) for C.glabrata. Isolates were considered to be different if the bandingpatterns differed by one, or more than one, readily detectableband.

Pulsed-field gel electrophoresis (PFGE) was performed asdescribed previously [32]. The electrophoretic karyotype (EK)was determined for C. albicans, C. tropicalis and C. glabrata. Inaddition, C. albicans and C. tropicalis were analysed byrestriction endonuclease analysis using SfiI 10 U ⁄mL andBssHII 4 U ⁄mL, respectively, followed by PFGE. Isolates wereconsidered to be identical if all readily detectable bands of oneisolate matched those of another. If not, the coefficients ofsimilarity were determined.

RESULTS

Epidemiological study

Between 1996 and 2000, there were 117 cases ofcandidaemia, but controls were found for only115; therefore, two cases were not included in theanalysis. Forty percent of the patients were fromintensive care units. The median time from the

Binelli et al. Nosocomial candiduria and candidaemia 539

� 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 12, 538–543

urine culture to the first positive blood culturewas 1 day (range )2–23 days) for cases and 1 day(range )2–18 days) for controls. The Candida spp.causing candidaemia and candiduria are summa-rised in Table 1. Variables associated statisticallywith candidaemia were candiduria and the pres-ence of fungi at other sites (Table 2). Both groupswere similar in terms of age, severity of clinicalcondition and days of hospitalisation (Table 3).

The variables included in the multivariateanalysis were candiduria, the presence of fungiat other sites, the use of antimicrobial drugs,neutropenia and cancer. The multivariate analysisonly showed an association of candidaemia withcandiduria and with previous antimicrobial use(Table 4). Deaths during hospitalisation and with-

in 30 days of discharge were significantly higherfor cases (51%) than for controls (37%) (p 0.02).

Microbiological study

There were 23 pairs of urine and blood isolatesfrom 23 patients; in ten (43%) cases, the speciesisolated from blood was different to that isolatedfrom urine (Table 5). The urine of one patientyielded two different species, namely C. glabrata(in the blood) and C. tropicalis. Molecular typingwas performed for 14 pairs of isolates.

Table 1. Causes of candidaemia and candiduria

Organism

Candidaemia

n (%)

Candiduria

in cases

n

Candiduria

in controls

n

Candida albicans 43 (37) 12 6Candida tropicalis 26 (23) 10 2Candida parapsilosis 21 (18) – –Candida glabrata 7 (6) 1 –Candida guillermondii 5 (4) – –Candida krusei 3 (3) – –Candida famata 2 (23) – –Candida kefyr 1 (1) – –Candida spp. 7 (6) 1 1Total 115 24 9

Table 2. Factors associated with candidaemia: univariateanalysis of categorical variables

Variable

n (%) of

OR 95% CI p value

Casesn = 115

Controlsn = 115

Male gender 68 (59) 70 (61) 0.89 0.45–1.74 0.86Candiduria 24 (21) 9 (8) 8.50 1.96–36.79 0.001Haematuriaa 63 (70) 59 (66) 1.07 0.52–2.22 1.00Proteinuriaa 51 (57) 48 (53) 1.06 0.54–2.10 1.00Care at day hospital 15 (13) 18 (16) 0.63 0.20–1.91 0.58Use of antimicrobial drugs 111 (97) 103 (90) 3.00 0.97–9.3 0.08Use of antifungal drugs 23 (20) 28 (24) 0.75 0.38–1.46 0.50Urinary catheter 71 (62) 71 (62) 1.00 0.54–1.86 0.87Central venous catheter 97 (84) 99 (86) 0.87 0.41–1.82 0.85Endotracheal tube 43 (37) 36 (31) 1.31 0.73–2.36 0.33Haemodialysis 12 (10) 12 (10) 1.00 0.32–3.10 0.77Peritoneal dialysis 3 (3) 1 (1) 3.00 0.31–28.84 0.62Total parenteral nutrition 21 (18) 19 (17) 1.17 0.54–2.52 0.84Surgical procedure 50 (43) 57 (50) 0.72 0.39–1.32 0.36Steroid use 44 (38) 40 (35) 1.19 0.67–2.13 0.66Chemotherapy 26 (23) 31 (27) 0.50 0.17–1.46 0.30Presence of underlying disease 113 (98) 114 (99) 0.50 0.05–5.51 1.00Cancer 41 (36) 31 (27) 2.00 0.94–4.27 0.10Diabetes mellitus 9 (8) 10 (9) 0.89 0.34–2.30 1.00Neutropenia 32 (28) 25 (22) 1.88 0.79–4.42 0.21Other diseases 114 (99) 114 (99) 1.00 0.06–15.99 0.48Fungi at other sites 25 (22) 10 (9) 2.67 1.24–5.74 0.01

aInformation available for 90 cases and 90 controls only.

Table 3. Factors associated with candidaemia: univariateanalysis of continuous variables

Variable Median Range p value

Age (years)Cases 46 7–89 0.18Controls 42 3–87

APACHE II score at hospitalisationCases 13 0–34 0.09Controls 12 0–52

APACHE II score on day of positive blood cultureCases 16 2–47 0.87Controls 16 1–39

Days of hospitalisation before positive blood cultureCases 19 0–127 0.33Controls 16 0–138

SD, standard deviation.

Table 4. Factors associated with candidaemia: results ofmultivariate analysis

Variable OR 95% CI p value

Candiduria 9.79 2.14–44.76 0.003Antimicrobial use 3.79 1.06–13.56 0.041

Table 5. Identification of pairs of Candida isolates fromblood and urine cultures

Patient number Blood culture Urine culture

1 C. albicans C. glabrata2 C. tropicalis C. tropicalis3 C. glabrata C. albicans4 C. glabrata C. tropicalis ⁄C. glabrata5 C. parapsilosis C. tropicalis6 C. tropicalis C. tropicalis7 C. albicans C. albicans8 C. albicans C. albicans9 C. albicans C. tropicalis

10 C. parapsilosis C. tropicalis11 C. tropicalis C. tropicalis12 C. tropicalis C. krusei13 C tropicalis C. glabrata14 C. tropicalis C. albicans15 C. tropicalis C. tropicalis16 C. albicans C. albicans17 C. tropicalis C. tropicalis18 C. albicans C. albicans19 C. albicans C. albicans20 C. albicans C. albicans21 C. tropicalis C. tropicalis22 C. albicans C. tropicalis23 C. tropicalis C. tropicalis

540 Clinical Microbiology and Infection, Volume 12 Number 6, June 2006


C. albicansTwelve isolates, comprising six pairs from urineand blood, generated four different DNA profileswhen analysed by RAPD using oligo 2, and threeprofiles when analysed by oligo 3. The pairs of C.albicans isolates were identical for each patient,regardless of which primer was used. EK analysisyielded three DNA profiles. The blood and urineisolates of each patient were also identical fol-lowing restriction endonuclease analysis.

C. tropicalisSeven pairs of blood and urine isolates werestudied by RAPD and PFGE. Primer P5 generatedfive different profiles; the profiles of the urine andblood samples were different in one pair. Fourdifferent profiles were produced using oligo 2; theblood and urine isolates were different foranother pair. The isolates could not be differen-tiated by EK analysis. In patients 15 and 23, thesimilarity coefficients for the blood and urineisolates were 71.5% and 76.5%, respectively,following restriction endonuclease analysis withBssHII, suggesting that the isolates might bedifferent.

C. glabrataPatient 4 yielded C. glabrata isolates from bloodand urine; the isolates were identical followingRAPD and PFGE analysis.

DISCUSSION

The significance of Candida isolated from urine isstill unclear. Candidaemia is a rare occurrence inhospitalised patients, and therefore a cohort studyto evaluate candiduria as a risk-factor for candid-aemia was not considered practical. Thus, a case-control study was chosen. In the present study,univariate analysis indicated a significant associ-ation of candidaemia with candiduria and theisolation of fungi at other sites. Multivariateanalysis showed an association between candid-aemia and candiduria and previous use of anti-microbial drugs. The OR for candiduria was 9.79.These findings suggest that patients with can-diduria have an increased risk for presenting withcandidaemia. It was not possible to define whe-ther candiduria was the source of candidaemia.Mortality was significantly higher among cases,but death can be associated with a number ofother factors, such as site of infection, underlying

conditions and adequate treatment, that were notevaluated in the present study. Thus, this resultshould be viewed with caution.

There are probably many factors involved in thegenesis of candidaemia, including extensive use ofantimicrobial agents and translocation from thedigestive tract [7]. Thus, the treatment of candidu-ria as a measure to prevent candidaemia [33,34]may not be an effective strategy. It is important todefine whether there is a causative associationbetween candidaemia and candiduria beforerecommending such extensive antifungal use.

In ten of 23 cases, the species of Candida isolatedfrom the blood and urine were different. Molecu-lar typing demonstrated that the paired isolatesfrom blood and urine were identical for each ofthe six patients with C. albicans. Among the sevenpatients with C. tropicalis, the paired isolates weredifferent for two patients. The blood and urineisolates of C. glabrata from one patient hadidentical profiles. In the present study, restrictionendonuclease analysis was more discriminatorythan RAPD, in accordance with previous findings[35,36]. Overall, among the 23 pairs of blood andurine isolates from 23 patients, the isolate in theblood was different from that in the urine for 52%of the patients. Therefore, the microbiologicaldata do not support the epidemiological associ-ation, which suggests that candiduria has thesame risk-factors as Candida bloodstream infec-tion, and that candidaemia may not be a directconsequence of candiduria. Thus the use ofempirical antifungal treatment in patients withcandiduria [33,34] could lead to unnecessaryantifungal prescription.

One of the limitations of the present study wasthe lack of routine surveillance for Candida atother sites, and this could explain why the‘presence of fungi at other sites’ variable wasnot significant by multivariate analysis. It was notpossible to ascertain whether patients withoutcultures were not colonised by fungi. Anotherlimitation was that there may have been mixedCandida urine infections that were not detected,although in one patient, two species of Candidawere isolated from the urine.

In studying nosocomial infections, the choice ofcontrols is often difficult. Using controls thatrepresented the source population was consid-ered, but it was felt that such individuals could beyounger and less ill than the cases. This couldresult in significant associations between candid-



aemia and previously well-studied factors, suchas age, days of hospitalisation, invasive devicesand severity of clinical condition [37], that mightovershadow the primary hypothesis (i.e., thepresence of candiduria as an independent risk-factor for candidaemia). Thus, control patientswith nosocomial bloodstream infections, whowould present a high prevalence of well-studiedrisk-factors for candidaemia, were chosen. For thesame reason, cases and controls were matchedbased on age. McCarthy and Giesecke [38] havediscussed the difficulty in choosing controls forstudies in infectious diseases, emphasising thatbias could occur because of differences in sur-veillance methods used to obtain cases andcontrols, and suggested that both groups shouldbe selected using the same method of surveillanceand the same infectious diagnosis, albeit causedby different microorganisms.

Established factors associated with systemicinfections, such as age, severity of condition(APACHE II score), previous days of hospitalisa-tion and the use of invasive devices, were similarin cases and controls. The choice of severely-illcontrols may have led to over-matching, andtherefore an underestimation of the ORs, but itwas considered that this was an adequate strategyto evaluate the association of candidaemia withcandiduria, since it suggests that the actual ORmay be > 9.8.

In conclusion, although candidaemia was asso-ciated strongly with candiduria in an epidemio-logical study, the microbiological study indicatedthat urine was not the main source of Candidabloodstream infection.

ACKNOWLEDGEMENTS

This study was supported by a doctoral grant from FAPESP(Fundacao de Amparo a Pesquisa do Estado de Sao Paulo),grant 98 ⁄ 13401–1.

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Binelli Et Al. 2006 Infecções Urinárias