oulu 2012 d 1163 university of oulu p.o.b. 7500 fi-90014
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Jarmo Salo
OULU 2012
D 1163
Jarmo Salo
LONG-TERM CONSEQUENCES AND PREVENTION OF URINARY TRACT INFECTIONSIN CHILDHOOD
UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU, FACULTY OF MEDICINE,INSTITUTE OF CLINICAL MEDICINE, DEPARTMENT OF PAEDIATRICS;OULU UNIVERSITY HOSPITAL
A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 1 1 6 3
JARMO SALO
LONG-TERM CONSEQUENCES AND PREVENTION OF URINARY TRACT INFECTIONS IN CHILDHOOD
Academic dissertation to be presented with the assent ofthe Doctoral Training Committee of Health andBiosciences of the University of Oulu for public defencein Auditorium 12 of the Department of Paediatrics, on 31August 2012, at 12 noon
UNIVERSITY OF OULU, OULU 2012
Copyright © 2012Acta Univ. Oul. D 1163, 2012
Supervised byProfessor Matti Uhari
Reviewed byProfessor Christer HolmbergDocent Harri Saxén
ISBN 978-951-42-9869-1 (Paperback)ISBN 978-951-42-9870-7 (PDF)
ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)
Cover DesignRaimo Ahonen
JUVENES PRINTTAMPERE 2012
Salo, Jarmo, Long-term consequences and prevention of urinary tract infections inchildhood. University of Oulu Graduate School; University of Oulu, Faculty of Medicine, Institute ofClinical Medicine, Department of Paediatrics; Oulu University Hospital, P.O. Box 5000, FI-90014 University of Oulu, FinlandActa Univ. Oul. D 1163, 2012Oulu, Finland
Abstract
Urinary tract infections (UTIs) are among the most common bacterial infections in childhood andhave a tendency to recur. The ability of uropathogens to form biofilm may be important in thepathogenesis of UTI. Although childhood UTIs are thought to increase the risk of chronic kidneydisease (CKD), evidence showing this association is scarce. Cranberry juice has been shown toprevent UTIs in women, but evidence of its efficacy in children is lacking. The aim of this workwas to evaluate the significance of biofilm formation for the clinical presentation of UTI, the long-term consequences of UTI in childhood and the efficacy, safety and acceptability of cranberryjuice in the prevention of UTIs in children.
The formation of biofilm in clinical samples was assessed in vitro with optical densitymeasurements and verified by scanning electron microscopy and confocal scanning lasermicroscopy. A systematic literature search on the association between childhood UTIs and CKDwas conducted, and data on patients with CKD treated or monitored at Oulu University Hospitalwere reviewed. The efficacy of cranberry juice in preventing recurrences of UTIs in children andits effects on the normal flora were evaluated with two randomized controlled trials.
About one third of the uropathogenic E. coli strains were capable of forming biofilm, and thestrains isolated from patients having pyelonephritis formed biofilm better than those from cystitiscases. We did not find any cases among the 1576 reviewed in the literature search or the 366 inour patient series who had structurally normal kidneys in their first kidney imaging and in whomchildhood UTIs could have been the cause of subsequent CKD. The aetiological fraction ofchildhood UTIs as a cause of CKD was at most 0.3%. The administering of cranberry juice did notreduce the number of children who experienced a recurrence of UTI, but it did reduce the numberof recurrences per person year at risk by 39% (0.25 vs. 0.41 episodes, 95% CI for difference -0.31to -0.01) and the number of days on antimicrobials per patient year by 34% (11.6 vs. 17.6 days,95% CI for difference -7 to -5). Cranberry juice was well accepted and tolerated by the childrenand did not cause harmful changes in the normal flora.
The ability of bacteria to persist and grow in a biofilm seems to be one of the significant factorsin the pathogenesis of UTIs. A child with normal kidneys is not at risk of developing CKD becauseof UTIs in childhood, so that imaging procedures after the first UTI can be focused on findingsevere urinary tract abnormalities. Taking into account the relatively innocent nature of childhoodUTIs, cranberry juice offers an alternative to antimicrobials for preventing UTIs in children. Themechanism of action of cranberry juice may be associated with biofilm formation byuropathogens.
Keywords: biofilms, child, chronic kidney insufficiency, cranberry, Escherichia coli,urinary tract infections
Salo, Jarmo, Lasten virtsatieinfektioiden pitkäaikaisseuraukset ja ennaltaehkäisy. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta, Kliinisenlääketieteen laitos, Lastentaudit; Oulun yliopistollinen sairaala, PL 5000, 90014 Oulun yliopistoActa Univ. Oul. D 1163, 2012Oulu
Tiivistelmä
Virtsatieinfektio (VTI) on yksi tavallisimpia lasten bakteeri-infektioita, ja sillä on taipumusuusiutua. Bakteerien biofilminmuodostuskyky voi olla merkittävä tekijä VTI:n synnyssä. Lap-suudessa sairastetun VTI:n ajatellaan lisäävän kroonisen munuaisten vajaatoiminnan riskiä,mikä ei kuitenkaan perustu tieteelliseen näyttöön. Karpalomehu ehkäisee aikuisten naistenVTI:n uusiutumisia, mutta sen tehoa lapsilla ei tiedetä. Tämän väitöskirjatyön tavoitteena oli sel-vittää bakteerien biofilminmuodostuskyvyn yhteyttä VTI:n kliiniseen kuvaan, lapsuudessa sai-rastetun VTI:n pitkäaikaisvaikutuksia sekä karpalomehun tehoa, turvallisuutta ja käyttökelpoi-suutta lasten VTI:n ehkäisyssä.
Kliinisistä virtsanäytteistä viljeltyjen E. coli –kantojen muodostama biofilmi mitattiin opti-sen tiheyden perusteella, ja elävien biofilmirakenteiden muodostuminen varmistettiin elektroni-mikroskooppi- ja konfokaalimikroskooppikuvauksilla. Lapsena sairastetun VTI:n yhteyttämunuaisten vajaatoimintaan tutkittiin systemaattisella kirjallisuuskatsauksella ja selvittämälläOYS:ssa munuaisten vajaatoiminnan vuoksi hoidossa olevien potilaiden munuaissairauden etio-logia. Karpalomehun tehoa, turvallisuutta ja käyttökelpoisuutta lasten VTI:n ehkäisyssä selvitet-tiin kahdella lumekontrolloidulla tutkimuksella.
Kolmasosa E. coli –kannoista muodosti biofilmiä, ja pyelonefriittipotilailta eristetyt kannatolivat parempia biofilminmuodostajia kuin kystiittipotilailta eristetyt. Kirjallisuuskatsauksen1576:n ja OYS:n 366:n tapauksen joukossa ei ollut potilaita, joilla olisi ollut ensimmäisessäkuvantamistutkimuksessa rakenteeltaan normaalit munuaiset ja lapsuuden VTI:t olisivat johta-neet munuaisten vajaatoimintaan. Lapsuuden VTI:n etiologinen fraktio munuaisten vajaatoimin-nan syynä oli teoriassakin korkeintaan 0.3 %. Karpalomehu ei vähentänyt uusintainfektion saa-neiden lasten määrää, mutta se vähensi uusintaepisodeja 39 % riskiaikaa kohti (0,25/vuosi vs.0,41/vuosi) ja antibioottipäiviä 34 % potilasvuotta kohti (11,6 vs. 17,6 päivää). Lapset joivat kar-palomehua mielellään, eikä sillä ollut haitallisia vaikutuksia normaaliflooraan.
Aiheuttajabakteerien biofilminmuodostuskyky vaikuttaa olevan merkittävä tekijä VTI:n pato-geneesissä. Lapsi, jolla on rakenteellisesti normaalit munuaiset, ei ole lapsuuden VTI:n vuoksiriskissä sairastua munuaisten vajaatoimintaan. Siten ensimmäisen VTI:n jälkeiset kuvantamistut-kimukset voidaan suunnata toteamaan vaikeat rakenteelliset poikkeavuudet. Koska lapsuudenVTI on suhteellisen vaaraton sairaus, karpalomehu on hyvä vaihtoehto VTI:n ehkäisyyn myöslapsilla. Biofilminmuodostuksen esto on sen yksi mahdollinen vaikutusmekanismi.
Asiasanat: biofilmi, Escherichia coli, karpalo, krooninen munuaisten vajaatoiminta,lapsi, virtsatieinfektiot
To my family
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Acknowledgements
The work for this thesis was carried out at the Department of Paediatrics, Oulu
University Hospital, Finland during the years 2001–2012. I am grateful to
Professor Mikko Hallman, MD, Chairman of the Department for creating an
inspiring atmosphere for scientific work during these years. I wish to express my
sincere gratitude to Docent Päivi Tapanainen, MD, the Head of the Division of
Paediatrics and Gynaecology, and to the late Professor Marjatta Lanning, MD,
former Chief Physician, for their support in combining clinical work and research
in the clinic.
I owe my deepest and warmest gratitude to my supervisor Professor Matti
Uhari, MD, for the dedicated, thorough and inspiring guidance and commendable
patience during the decade of my paediatric research. I am also grateful for his
valuable advice on computers, software and operating systems.
I am grateful to the official referees Professor Christer Holmberg, MD, and
Docent Harri Saxen, MD, for constructive comments on the manuscript.
I present my warmest thanks to co-authors Docent Tero Kontiokari, MD,
Docent Terhi Tapiainen, MD, Tytti Pokka, MSc, Professor Matti Korppi, MD,
Docent Merja Helminen, MD, Tea Nieminen, MD, Docent Erkki Eerola, MD, and
Jenny Sevander, MD, for adding their expertise to this work. I am especially
grateful to Docent Risto Ikäheimo, MD, Docent Irma Ikäheimo, PhD, and Docent
Markku Koskela, MD, without whom this thesis would not have been possible. I
also thank Salli Saulio, RN, for dedicated and competent work as study nurse in
our cranberry studies.
The members of our research team, InTeam, deserve my gratitude for
enthusiastic conversations and direct feedback. In addition to lively discussions
on science and medicine, our weekly sessions have entertainment value worth
selling tickets to.
I am sincerely grateful to all the children and their families, patients and the
day-care centers for participating in this work. I also thank the staff of the
participating hospitals for recruiting the children.
I thank Malcolm Hicks, MA, for revising the English language of the original
papers and of this thesis, and Juha Turtinen, MSc, the secretaries in our
department Marjatta Paloheimo and Aila Kokko, and librarian Maija Veikkola for
their friendly assistance.
All my colleagues and friends at the Department of Paediatrics are
appreciated for their friendship and support during these years. As a former
10
chairman of SAK I am delighted to see that the posse is as active as ever and the
quality of singing has remained constant.
I feel myself privileged for having loyal friends whose support and help for
my family and me cannot be put into words in any publication. I am grateful for
the camaraderie and sophisticated company they have provided. I send my special
thanks to brothers Young for their inspiring art.
I wish to express my loving thanks to my mother Hillevi Salo and my father
Kari Salo for providing constant and unconditional love, support and
encouragement throughout my life. I would also like to show my gratitude to my
godmother Kaisu Kiiskinen and Kirsti Kiiskinen who have always been there for
me.
Finally, I dedicate this thesis to my wife Anu and our three sons Juho, Eemi
and Aarni who share my interest in life and its wonders. You are everything for
me.
This work has been supported financially by the Juho Vainio Foundation, the
Paulo Foundation, the Päivikki and Sakari Sohlberg Foundation, the Foundation
for Paediatric Research, the Finnish Kidney Foundation and Ocean Spray
Cranberries Inc, which all are gratefully acknowledged.
Oulu, June 2012 Jarmo Salo
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Abbreviations
ABU Asymptomatic bacteruria
CFU Colony forming unit
CI Confidence interval
CKD Chronic kidney disease
CL Confidence limit
CP Chronic pyelonephritis
CRP C-reactive protein
CSLM Confocal scanning laser microscopy
DMSA Dimercaptosuccinic acid
FSGS Focal segmental glomerulosclerosis
GLC Gas liquid chromatography
IVU Intravenous urography
OD Optical density
PAC Proanthocyanidins
PCT Procalcitonin
PYR Person years at risk
RCT Randomized controlled trial
RRT Renal replacement therapy
SD Standard deviation
SEM Scanning electron microscopy
SPA Suprapubic aspiration
UPEC Uropathogenic Escherichia coli UTI Urinary tract infection
VUR Vesico-ureteral reflux
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List of original publications
This thesis is based on the following publications, which are referred to in the text
by their Roman numerals.
I Salo J, Sevander J, Tapiainen T, Ikäheimo I, Pokka T, Koskela M & Uhari M (2009) Biofilm formation by Escherichia coli isolated from patients with urinary tract infections. Clinical Nephrology 71: 501–7
II Salo J, Ikäheimo R, Tapiainen T & Uhari M (2011) Childhood urinary tract infections as a cause of chronic kidney disease. Pediatrics 128: 840–7
III Salo J, Uhari M, Helminen M, Korppi M, Nieminen T, Pokka T & Kontiokari T (2012) Cranberry Juice for the Prevention of Recurrences of Urinary Tract Infections in Children: A Randomized Placebo-Controlled Trial. Clin Infect Dis 54: 340–6
IV Kontiokari T, Salo J, Eerola E & Uhari M (2005) Cranberry juice and bacterial colonization in children – a placebo-controlled randomized trial. Clin Nutr 24: 1065–72
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Contents
Abstract
Tiivistelmä
Acknowledgements 9 Abbreviations 11 List of original publications 13 Contents 15 1 Introduction 17 2 Review of the literature 19
2.1 Urinary tract infections in childhood ...................................................... 19 2.1.1 Definitions .................................................................................... 19 2.1.2 Epidemiology ............................................................................... 19 2.1.3 Symptoms and diagnosis .............................................................. 20 2.1.4 Treatment ...................................................................................... 24 2.1.5 Imaging of the urinary tract after the first UTI ............................. 24
2.2 Pathogenesis of urinary tract infections in childhood and risk
factors ...................................................................................................... 25 2.2.1 Pathogens and virulence factors ................................................... 26 2.2.2 Anatomical factors ....................................................................... 28 2.2.3 Functional factors ......................................................................... 28 2.2.4 Other risk factors .......................................................................... 29
2.3 Long-term consequences of urinary tract infections in childhood .......... 30 2.3.1 Chronic kidney disease ................................................................. 30 2.3.2 Other consequences ...................................................................... 31
2.4 Prevention of urinary tract infections in childhood ................................. 31 2.4.1 Antimicrobials .............................................................................. 31 2.4.2 Dietary factors .............................................................................. 32 2.4.3 Cranberry ...................................................................................... 32 2.4.4 Other preventive measures ........................................................... 34
3 Aims of the research 37 4 Biofilm formation by uropathogens as an aspect of pathogenesis (I) 39
4.1 Subjects ................................................................................................... 39 4.2 Methods ................................................................................................... 40 4.3 Results ..................................................................................................... 43
5 Long-term consequences of urinary tract infections in childhood (II) 45 5.1 Methods ................................................................................................... 45
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5.2 Results ..................................................................................................... 49 6 Cranberry juice for the prevention of urinary tract infections
(III and IV) 57 6.1 Subjects ................................................................................................... 57 6.2 Methods ................................................................................................... 60 6.3 Results ..................................................................................................... 65
7 Discussion 73 7.1 Biofilm formation by uropathogens as an aspect of
pathogenesis (I) ....................................................................................... 73 7.2 Long-term consequences of urinary tract infections in
childhood (II) .......................................................................................... 74 7.3 Cranberry juice for the prevention of urinary tract infections
(III and IV) .............................................................................................. 77 8 Conclusions 83 References 85 Original publications 103
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1 Introduction
Urinary tract infections (UTIs) are among the most common bacterial infections
in childhood, and recurrences are common (Winberg et al. 1974, Uhari &
Nuutinen 1988, Mårild & Jodal 1998, Nuutinen & Uhari 2001, Craig et al. 2009).
Dietary factors are associated with susceptibility to UTIs but the exact
pathomechanisms leading to UTI and its recurrences in children are not well
known (Kontiokari et al. 2004). Several Escherichia coli (E. coli) strains are
capable of forming biofilms (Reisner et al. 2006, Hancock et al. 2007), but the
clinical significance of bacterial biofilms for the level of UTI and for its
recurrences is largely unknown (Kanamaru et al. 2006, Soto et al. 2007).
Childhood UTIs cause kidney scarring and are said to lead to impaired kidney
function later, especially in the presence of vesicoureteral reflux (VUR)
(Jakobsson et al. 1999b, Lahdes-Vasama et al. 2006, Swerkersson et al. 2007).
Children are therefore normally subjected to radiological imaging after the first
UTI and long-term antibiotic prophylaxis if grade III to V VUR is found. The
most severe consequence of childhood UTIs has been thought to be chronic
kidney disease (CKD), but evidence for an association between childhood UTIs,
renal damage and later CKD in children with structurally normal kidneys is scarce
(Hellerstein 2000). Accurate diagnosis is the basis of all actions taken after UTI,
so it is essential that the diagnosis should be made correctly and using adequate
methods.
Many children with recurrent UTI need antimicrobial prophylaxis for years,
which is not without problems (Uhari et al. 1996b, Williams et al. 2001). Adverse
reactions and cessation of therapy are common during this long-term antibiotic
regimen, and there is an increasing problem of bacterial resistance (Uhari et al. 1996b). Cranberry products appear to be able to prevent UTI recurrences in adult
women, but evidence for their efficacy in children is lacking (Blatherwick 1914,
Avorn et al. 1994, Schlager et al. 1999, Kontiokari et al. 2001, Stothers 2002,
Jepson et al. 2004, Wing et al. 2008). The acceptability of cranberry juice has
been questioned as well, and the broad in vitro antimicrobial spectrum of
cranberry raises concerns about the possibility of adverse events during
continuous use (Jepson et al. 2004).
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2 Review of the literature
2.1 Urinary tract infections in childhood
2.1.1 Definitions
A urinary tract infection (UTI) is defined as the presence of microbial pathogens
within one or more structures in the urinary system, including the urethra, bladder
and kidneys, in a symptomatic patient. Cystitis is an infection of the lower urinary
tract (urethra, urinary bladder), pyelonephritis an infection of the upper urinary
tract (ureter, kidneys) and pyelitis an infection of the renal pelvis. Asymptomatic
bacteruria is defined as the significant growth of bacteria in urine without
symptoms of UTI. A diagnosis of urosepsis is recorded if the patient has clinical
sepsis and bacteraemia that have originated from bacteruria caused by bacteria
capable of inducing inflammation in the urinary tract. UTI in patients with a
normal urinary tract is regarded as uncomplicated, whereas complicated UTI is
associated with urinary tract abnormalities, catheters or operations. Recurrent
UTIs are classified as relapses or reinfections, relapses being caused by the same
bacterial strain as the initial episode and occurring within a few months of the
cessation of therapy, while reinfections are caused by a new bacterial strain.
2.1.2 Epidemiology
About 2–8% of children have at least one symptomatic UTI before puberty
(Winberg et al. 1974, Hellström et al. 1991, Mårild & Jodal 1998, Conway et al. 2007, Shaikh et al. 2008), and almost one percent have UTI before the age of six
(Conway et al. 2007). The occurrence is highest in infancy, when about 2/3 of the
UTI patients are boys, after which time the majority of patients are girls
(Ginsburg & McCracken 1982, Jakobsson et al. 1999a, Foxman 2003, Conway et al. 2007). UTI is a diagnostic challenge, especially among young children. About
5% of infants with a fever of unknown origin at admission have UTI, and about
5% of children less than two years of age with UTI have bacteraemia (Hoberman
et al. 1993a, Hoberman et al. 1999). UTI is cause of 15% of all cases of fever of
unknown origin in girls under two years of age (Shaw et al. 1998). After the first
episode, 10–30% of patients experience a recurrence or reinfection, mostly within
six months of the initial UTI episode (Uhari et al. 1996b, Nuutinen & Uhari 2001,
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Jantunen et al. 2002, Mingin et al. 2004b, Conway et al. 2007, Craig et al. 2009).
The incidence of UTI in childhood is 0.2%-0.3% per year and has been
decreasing since the 1970s, presumably in response to changes in diet, the quality
of napkins and child-care practices in general (Dickinson 1979, Uhari & Nuutinen
1988). The incidence of UTI and its diagnostic rate differ between populations
and centres in addition to true differences in incidence, partly because of
variations in diagnostic methods, diagnostic criteria, UTI awareness and the
organisation of health-care systems (Jakobsson et al. 1999a).
2.1.3 Symptoms and diagnosis
Accurate diagnosis is the basis of all reactions to UTI, so it is essential that the
diagnosis should be correct and made using adequate methods. The diagnosis of
UTI is based on clinical signs and/or symptoms, urinalysis (leucocytes, nitrite)
and the presence of a significant growth of bacteria in the urine. The symptoms
depend on the level of infection and the age of the patient, but typical
manifestations include fever, dysuria, frequency, urgency, enuresis, abdominal
pain, flank back pain and incontinence. The symptoms are less specific in infants
than in older children, however. Urosepsis and pyelonephritis cause similar
symptoms and are clinically indistinguishable from each other (Honkinen et al. 2000). Decision rules based on symptoms and patient characteristics have been
developed and studied in order to select high-risk patients for urinalysis and
treatment and low-risk patients for follow-up (Gorelick et al. 2003, Shaikh et al. 2007). In girls younger than two years, for example, a fever of 39.0 °C or more in
the absence of any other potential cause and fever for two days or more entail
with a high risk of UTI (Gorelick et al. 2003). Children with UTI cannot be
distinguished from non-infected children on the basis of symptoms alone,
however (Pylkkanen et al. 1979, Shaikh et al. 2007).
A urine collection pad or bag are reliable and easy methods for taking
samples for urinalysis, and in the case of young children urinalysis can be
performed on a urine bag or cushion/pad sample for use in assessing the
likelihood of UTI (Liaw et al. 2000, Macfarlane et al. 2005, McGillivray et al. 2005, Schroeder et al. 2005, American Academy of Pediatrics 2011). A urine
collection pad should be changed every 30 minutes in order to reduce the
possibility of a false positive result (Rao et al. 2004). Bag samples are especial
prone to contamination of the urine culture (Al-Orifi et al. 2000), and thus the
diagnosis of UTI children who are unable to control their bladder should be
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established from a sample collected by suprapubic aspiration (SPA), or by
transurethral catheterization if the SPA method is unsuccessful (Pylkkanen et al. 1979, American Academy of Pediatrics 2011). Beyond infancy a midstream urine
sample can be taken, whereupon the number of false positive results can be
reduced by positioning a sterile cup in the front part of the urinal (Huttunen et al. 1970).
SPA is the gold standard for taking samples for urine culture (American
Academy of Pediatrics 2011). It is safe and complications are rare, but timing of
the procedure is not always easy because of irregular voiding by infants (Polnay
et al. 1975, O'Callaghan & McDougall 1987, Moustaki et al. 2007). The success
rate of SPA can be improved by real-time ultrasound guidance (O'Callaghan &
McDougall 1987, Chu et al. 2002). A urine sample taken by SPA can be stored
and cultivated later when necessary without risk of contamination, because any
growth of bacteria in such a sample is significant (Bailey & Little 1969). In
children aged less than two months SPA is a more painful way of taking urine
samples than catheterization (Kozer et al. 2006). In the classic study by Pryles et al. the sensitivity and specificity of transurethral catheterization for diagnosing
UTI were excellent by comparison with SPA, but in a recent study more than 10%
of the catheterized urine samples were contaminated, and the rate of
contamination was especially high in uncircumcised boys (Pryles et al. 1959,
Wingerter & Bachur 2011).
The rapid screening tests available for UTI are based on dipstick tests, gram
staining, microscopic analysis or various combinations of these. Their
sensitivities and specificities for UTI depend on the selection of the patients for
testing, the methods by which the urine samples are taken, the processing of the
samples and the cut-off points used (Gorelick & Shaw 1999, Huicho et al. 2002).
Hoberman et al. found that the presence of more than 10 leukocytes/ml had a
sensitivity of 91% and a false positivity rate of 3.4% for identifying significant
growth in cultures of urine samples taken by catheter, while the presence of either
leucocytes or bacteria in an enhanced urinalysis had a sensitivity of 95% for
finding positive urine cultures (Hoberman et al. 1994, Hoberman et al. 1996).
When a positive urinalysis is defined as entailing the presence of both bacteruria
and pyuria, enhanced urinalysis (using an uncentrifuged specimen) has a better
sensitivity and positive predictive value (85% and 93%, respectively) than the
standard analysis (66% and 81%, respectively), and both of them have a
specificity and negative predictive value of about 99% (Hoberman et al. 1993b).
The sensitivity of urinalysis is better among older children than in children under
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two years of age (Doley & Nelligan 2003). Standard urinalysis has a sensitivity of
82% and a specificity of 92% for diagnosing UTI in febrile children aged less
than two years (Bachur & Harper 2001), while the sensitivity of pyuria for
diagnosing UTI in samples taken by transurethral catheterization in febrile infants
was 30% (>10 WBC/high-power field) to 80% (any WBC/HPF) and specificity
47% (any WBC/HPF) to 99% (>10 WBC/HPF) (Hoberman et al. 1993a). The
sensitivity of urinalysis is better when the samples are taken with urine bags than
with a catheter but the specificity is poorer (McGillivray et al. 2005).
Most of the bacteria causing UTI can reduce dietary nitrate to nitrite, a
phenomenon that can be measured and utilized diagnostically. The sensitivity of
the nitrite test for symptomatic UTI is less than 50%, however, although the
specificity is 99% (Powell et al. 1987, Hoberman et al. 1994). The sensitivity of
the nitrite test is higher for asymptomatic bacteruria (ABU) than for UTI (Kunin
& DeGroot 1977, Powell et al. 1987). Inclusion of the nitrite test in urinalysis
enhances the sensitivity of the latter for UTI, and when both leukocyte esterase
and nitrite tests are used the sensitivity is more than 90% (Conway et al. 2007).
Urine is a good growth medium for bacteria, and bacteria can grow to a
maximum density of about 108/ml in a few hours in sterile urine (Kass 1956,
Wheldon & Slack 1977). On the other hand, bacteria from the distal urethra and
perineum also grow well and rapidly in urine (Wheldon & Slack 1977), so that in
order to avoid contamination, the sample should be cultured immediately or after
only brief storage at 5 °C. When diagnosing UTI it is important to distinguish
between contamination and true bacteruria. In the classic study by Kass et al. 95% of the women with pyelonephritis had a bacterial growth of 105 or more per
ml in a urine sample taken by catheter, and 3% had 104–105 bacteria per ml (Kass
1956). In addition, 80% of the women with a growth of 105 or more bacteria per
ml had a positive gram stain for their urine (Kass 1956). This would imply that
the definition of bacteruria in adults is 105 or more colony forming units (CFU) of
a single bacterial strain in a clean voided urine sample. The result of a urine
culture is more reliable if the urine has remained in the bladder for at least four
hours and when two separate samples are taken. Based on the study by Kass et al., significant growth in paediatric urine samples is usually defined as the growth of
a single bacterial strain to 105 or more CFU/ml in a midstream urine sample, any
growth in a suprapubic bladder aspirate sample, or growth of at least 50 000
CFU/ml in a catheter sample (Kass 1956, Hoberman et al. 1994, American
Academy of Pediatrics 2011). This definition is not based on firm evidence,
however, and it should be noted that as about 20% infants with true UTI have
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bacterial counts of less than 105 CFU/ml, the use of SPA or catheterization for
sampling is important in order to avoid both the under-diagnosing and over-
diagnosing of UTI (Hansson et al. 1998).
Cystitis has no long-term consequences, but pyelonephritis is thought to
cause kidney scarring, which may lead to chronic kidney disease (CKD) and
hypertension. Consequently, pyelonephritis is considered to require a more active
approach to diagnosis, therapy and follow-up than lower urinary tract infections.
There is thus a need for a reliable method for distinguishing between infections of
the upper and lower urinary tract. Dimercaptosuccinic acid (DMSA) scanning is
commonly used to determine the level of UTI, and power Doppler
ultrasonography correlates well with DMSA in diagnosing the level of UTI
(sensitivity and specificity both about 90%) (Jakobsson et al. 1992, Halevy et al. 2004). The significance of the findings obtained from a DMSA scan is somewhat
unclear, however (consequences of UTI vs. congenital abnormalities) and may be
dependent on the age of the patients (Ilyas et al. 2002, Shaikh et al. 2010). There
are no direct methods for determining the level of UTI in clinical practise.
Pyelonephritis is usually defined as UTI associated with a fever of over 38.5 ºC
and/or a C-reactive protein value greater than 40 mg/l (Jodal et al. 1975).
Procalcitonin (PCT) may be better than C-reactive protein (CRP) for diagnosing
the level of UTI (Gervaix et al. 2001, Pecile et al. 2004). Benador et al. (1998)
reported the sensitivity and specificity of PCT for finding renal lesions in DMSA
to be 70% and 83%, respectively, whereas CRP had sensitivity of 100% but a
specificity of 26% (Benador et al. 1998). Patients are diagnosed as having
urosepsis if they have UTI and a blood culture positive for the same pathogen.
Such patients have higher CRP, but the range is wide, so that CRP is of little use
for differential diagnosis between pyelonephritis and urosepsis (Honkinen et al. 2000).
ABU has no marked consequences, and screening and treating bacteruria
does not help prevent either UTI episodes or renal scarring (Kemper & Avner
1992, Harding et al. 2002). Furthermore, screening of ABU would result in a
great number of false positive tests (Kemper & Avner 1992, Kramer et al. 1994).
Thus ABU should not be treated or screened for in children (American Academy
of Pediatrics 2011).
24
2.1.4 Treatment
Standard antibiotic therapy aimed at E. coli is very effective, and fails to eradicate
bacteruria in only 1–3% of UTI episodes in children (Winberg et al. 1974, Craig
et al. 1998). Short-term oral antibiotic therapy (2–4 days) is as effective as the
standard therapy (7–10 days) for treating cystitis in children (Michael et al. 2012),
and a single dose of trimethoprim or trimethoprim-sulphamethoxazole is as
effective as a 5 or 7-day course for this same purpose, but the risk of bacteruria
after cessation of the therapy is higher (Pitt et al. 1982, Nolan et al. 1989, Lidefelt
et al. 1991). Oral and intravenous treatment are equally as effective for
pyelonephritis in children, and oral therapy is a safe and effective alternative
when hospitalization is not necessary (Hodson et al. 2007). The recommended
duration of therapy is 10–14 days, this is not based on firm evidence (Hodson et al. 2007). Oral and initial intravenous therapies are also equally effective for
treating UTIs in 1–24 -month-old children provided that the absorption of
antibiotics in the gastrointestinal tract is sufficient (Hoberman et al. 1999). The
urine cultures become sterile at a similar time (24 h), and there is no difference in
the reinfection rate (5%–9%) or in the extent of scarring (8%–9% of the renal
parenchyma). Even infants under two months of age with febrile UTI can be
treated safely with brief hospitalization and close follow-up without any marked
risk of complications, provided they are not clinically ill on admission
(Schnadower et al. 2010). Recent antimicrobial therapy increases the risk of UTI
caused by a resistant pathogen (Paschke et al. 2010). ABU should only be treated
in pregnant women (Nicolle et al. 2005, American Academy of Pediatrics 2011).
2.1.5 Imaging of the urinary tract after the first UTI
According to widespread recommendations, the urinary tract should be routinely
imaged after the first UTI in childhood, but, again, these recommendations are not
based on firm evidence (Dick & Feldman 1996, American Academy of Pediatrics
2011). The need for invasive radiological examinations after UTI has been
questioned, and the number of voiding mictiocystographies etc. performed in
clinical practise has been decreasing during the last two decades (South 2009,
Venhola & Uhari 2009). Ultrasonography performed after the first UTI in
childhood is a sufficient method for detecting clinically significant abnormalities
in the urinary tract, such as obstructive disorders (Jahnukainen et al. 2006,
American Academy of Pediatrics 2011, Hannula et al. 2011, Pennesi et al. 2012).
25
Over 80% of all major congenital malformations and minor abnormalities of the
urinary tract are found in antenatal ultrasonography (Grandjean et al. 1999). In
addition, some authors have maintained that the findings in ultrasonography do
not alter the management of a case after the first UTI (Hoberman et al. 2003,
Zamir et al. 2004, Miron et al. 2007, Montini et al. 2009). It should be noted that
many functional factors, e.g. constipation, may cause reversible abnormalities
identifiable in imaging examinations (Dohil et al. 1994). DMSA scanning is
considered to be a reliable method for detecting or ruling out abnormalities in the
kidney parenchyma, and some experts recommend it for detecting renal scars
after an acute infection, but the clinical significance of these findings is not well
established (Jakobsson et al. 1992, Stokland et al. 1999, Ilyas et al. 2002, Montini
et al. 2009, Shaikh et al. 2010).
2.2 Pathogenesis of urinary tract infections in childhood and risk factors
UTI is almost always an ascending infection, so that the bacterial strain causing it
can be found in the patient’s faeces (Jantunen et al. 2001a). In order to colonize
the urinary tract and cause infection, bacteria from the faecal or periurethral flora
must ascend to the bladder, survive the defence mechanisms, adhere to the
uroepithelial cells and proliferate (Hooton 2000, Finer & Landau 2004). In spite
of the excretion of bactericidic compounds, bacteria grow well in urine.
Uroepithelial cells have several defence mechanisms against bacteria (physical
barrier, defensins, cytokines, chemokines, recruitment of neutrophils and other
leucocytes) which are activated by the attachment of bacteria (Jahnukainen et al. 2005), but in addition to protecting the urinary tract, these mechanisms also cause
damage to the renal parenchyma and disturbances in renal function through
inflammatory responses (Jahnukainen et al. 2005, Gil-Ruiz et al. 2011). If the
host defence mechanisms and inflammatory response are not activated, invasion
of the urinary tract by bacteria may lead to asymptomatic bacteruria.
After becoming attached to the uroepithelium, uropathogens may invade the
uroepithelial cells and form biofilm-like structures inside them (Reid et al. 1993,
Mulvey et al. 2001, Anderson et al. 2003). Most recurrences of UTI in infants are
caused by the same bacterial strain as the initial episode, and reinfections are in
the minority (Jantunen et al. 2002). On the other hand, recurrences caused by an
E. coli strain identical to that implicated in the first UTI occur earlier than those
caused by a different strain (Foxman et al. 2000). Thus it seems that a significant
26
proportion of recurrent UTIs may be of endogenous origin (Jantunen et al. 2002).
The finding of biofilm-like structures in the uroepithelium supports this theory
(Reid 1994, Mulvey et al. 2001, Anderson et al. 2003).
2.2.1 Pathogens and virulence factors
Escherichia coli (E. coli) is the leading pathogen causing UTIs in childhood,
being implicated in about 80% of cases (Honkinen et al. 1999, Conway et al. 2007, Craig et al. 2009). Also, UTI episodes caused by E. coli are more likely to
recur than those caused by other bacteria (Foxman et al. 2000). Bacteria with low
virulence have better chances of causing UTI in children who have functional or
anatomical abnormalities in the urinary tract (Honkinen et al. 1999), and if UTI is
caused by a less virulent pathogen, the probability of urinary tract abnormalities
will be higher than when UTI is caused by uropathogenic E. coli (UPEC)
(Jantunen et al. 2001b).
The ability of bacteria to attach to the uroepithelium and cause UTI is
determined by virulence factors such as adhesins, toxins, the capsule, siderophore
and lipopolysaccharides (Kaper et al. 2004). The adhesins associated with UPEC
strains include type 1 pili, P pili, S pili and Dr adhesins (Kaper et al. 2004), the
blocks of genes encoding these virulence factors being situated in pathogenicity
islands (Hacker & Kaper 2000). The two most important adhesins of E. coli are
type 1 fimbria (or pili) and P fimbria (or pili). UPEC adheres to the uroplakin in
uroepithelial cells by means of type 1 fimbriae (Connell et al. 1996, Mulvey et al. 1998), while P fimbriae are associated with acute pyelonephritis (Roberts et al. 1994). Most UPEC strains have the ability to express both adhesins in response to
growth conditions (Xia et al. 2000). Flagella are responsible for the motility of
UPEC and are important for colonization of the urinary tract (Lane et al. 2005,
Lane et al. 2007). In addition to colonization, type 1 fimbriae also have an
essential function in the process of intracellular biofilm formation (Pratt & Kolter
1998, Mulvey et al. 2001, Wright et al. 2007). Bacteria causing ABU can persist
in the urinary tract without adhesins and resist host responses by virtue of their
high growth rate, and can even protect the subject from UTI by out-competing the
uropathogenic bacteria for colonization of the urinary tract (Roos et al. 2006).
This mechanism is probably associated with their better iron acquisition, achieved
through upregulation of the genes involved (Roos et al. 2006). The ability of
bacteria to form a biofilm is also important in the colonization process (Jefferson
27
2004), the E. coli strains causing ABU being better biofilm formers than the
UPEC strains (Hancock et al. 2007).
Bacterial biofilms are sessile bacterial communities attached to a surface and
forming organized structures surrounded by an exopolysaccharide matrix
(Costerton et al. 1999, Webb et al. 2003, Branda et al. 2005, Tenke et al. 2006).
Biofilms protect bacteria from harmful conditions and host defences, and the
bacteria in a biofilm benefit from co-operation, as they are able to act together in
the manner of a multi-cellular organism. Also a biofilm can be a source of new
bacterial colonies (Jefferson 2004). Living bacteria are constantly being released
from a biofilm, which can therefore give rise to infection (Figure 1). Several E. coli strains are capable of forming biofilms, and these have been found in
catheters, the prostate and kidney stones (Adams & McLean 1999, Danese et al. 2000a, Danese et al. 2000b, Banning et al. 2003, Wakimoto et al. 2004, Tenke et al. 2006, Hancock et al. 2007). Bacteria have also been found in the
uroepithelium of patients with a previous history of UTI (Elliott et al. 1985).
Bacteria in intracellular biofilms are safe from antibiotics because of inadequate
penetration and other mechanisms associated with the architecture of a biofilm,
the low metabolic rate of bacteria in a biofilm, etc. (Stewart & Costerton 2001,
Zheng & Stewart 2002). Some antibiotics, such as aminoglycosides, may even
induce biofilm formation (Hoffman et al. 2005). There is evidence that the
elimination of bacteria in a biofilm can be induced with agents derived from
plants, such as forskolin (Bishop et al. 2007).
28
Fig. 1. Stages of biofilm formation: A) initial attachment of planktonic bacteria, B) irreversible adhesion and formation of the biofilm matrix, C) growth and multiplication of bacteria in the biofilm, D) a mature biofilm, and E) dispersion of the planktonic bacteria and formation of new colonies.
2.2.2 Anatomical factors
Urinary tract abnormalities affecting the flow of urine and emptying of the
bladder increase the risk of UTI. Male circumcision reduces the risk of UTI
substantially (odds ratio 0.13, 95% CI 0.08 to 0.20, p<0.001) (Singh-Grewal et al. 2005), so that the reported incidence of UTI in infancy is greater in non-
circumcised boys than in circumcised boys (1–2% vs. 0.1–0.2%) (Wiswell &
Roscelli 1986, Schoen et al. 2000). Also, children with UTIs caused by pathogens
other than E. coli have more functional or anatomical abnormalities in the urinary
tract than those with UTIs caused by E. coli (Honkinen et al. 1999). Urinary tract
obstructions and high-grade reflux are more common among patients with
urosepsis than with pyelonephritis (9% vs. 1% and 40% vs. 22%, respectively)
(Honkinen et al. 2000).
2.2.3 Functional factors
Dysfunctional elimination syndrome (bladder and bowel incontinence and
withholding) diagnosed after the age of two increases the risk of recurrent UTIs
(Shaikh et al. 2003, Mingin et al. 2004a), and it is this dysfunctional elimination
that causes children aged 3 to 5 years to have a higher risk of recurrent UTI than
29
either younger and older children (Conway et al. 2007). Recurrent UTIs are more
common among children who have daytime wetting and high voiding frequency
(Bakker et al. 2004). Also, the starting of potty training after the age of 18 months
and excessively active potty training are associated with a higher risk of recurrent
UTIs (Bakker et al. 2004). Voiding dysfunction, e.g. detrusor instability or any
other factor affecting the flow of urine, will increase the risk of UTI (Hellerstein
& Linebarger 2003), the most common cause of detrusor instability being
constipation (Hellerstein & Linebarger 2003). Although there is an association
between bladder dysfunction and UTIs, it is not clear which comes first
(Hellström et al. 1991). Constipation is more common in children with UTI than
in controls (Dohil et al. 1994, Blethyn et al. 1995), and it is known to increase the
risk of UTIs, whereas its treatment prevents the recurrence of UTIs (Neumann et al. 1973, Loening-Baucke 1997). Constipation increases the residue after
micturiation, causes functional obstruction and affects the flow of urine
(Neumann et al. 1973, Dohil et al. 1994, Blethyn et al. 1995, Loening-Baucke
1997).
The concept of vesicoureteral reflux (VUR) as a cause of renal damage was
based on the findings among paraplegic adult patients (Hutch 1952).
Vesicoureteral reflux (VUR) is found in 20–40% of children after the first UTI
(Dick & Feldman 1996, Honkinen et al. 1999, Hannula et al. 2010, Shaikh et al. 2010), and contrary to the earlier understanding, it seems to be common among
normal children and to occur at similar rates among children with or without UTI
(Köllerman & Ludwig 1967, Ataei et al. 2004, Hannula et al. 2010). VUR of
grades 1 to 3 has no effect on the risk of UTI recurrence but grades 4 and 5 carry
a higher risk (Nuutinen & Uhari 2001, Conway et al. 2007). High-grade VUR is
more common among patients with urosepsis than with pyelonephritis (Honkinen
et al. 2000). VUR alone has no severe long-term consequences and is neither a
necessary nor a sufficient cause for renal scars (Smellie et al. 1998, Shaikh et al. 2010). Operative treatment is effective in correcting VUR, but does not reduce the
incidence of UTI, nor does it improve the prognosis in the case of children (Mor
et al. 2003, Venhola et al. 2006, Hannula et al. 2010).
2.2.4 Other risk factors
Dietary factors are associated with susceptibility to UTIs (Kontiokari et al. 2003,
Kontiokari et al. 2004). Poor fluid intake and even mild hydration are risk factors,
but the evidence for this association is not consistent (Beetz 2003, Gray &
30
Krissovich 2003). There seems to be a genetic predisposition to UTIs (Hopkins et al. 1999b, Scholes et al. 2000, Zaffanello et al. 2010), with a history of UTI in the
mother (odds ratio 2.3, 95% CI, 1.5–3.7) and the first UTI in childhood (odds
ratio 3.9, 95% CI, 1.9–8.0) acting as risk factors for recurrent UTIs in early
adulthood (Scholes et al. 2000). The attachment of E. coli to uroepithelial cells is
determined by the receptors of the epithelial cells, and is higher in the
uroepithelial cells of ABH blood group antigen non-secretors than those of
secretors (Lomberg et al. 1986). However, neither the ABO phenotype nor the
Lewis blood cell antigen phenotype entails a risk of recurrent UTIs (Hopkins et al. 1998). The risk of UTI seems not to be related to the use of nappies, the type of
nappy used or other nursing habits (Nuutinen et al. 1996).
2.3 Long-term consequences of urinary tract infections in childhood
Childhood UTIs cause substantial morbidity and are thought to increase the risk
of chronic kidney disease and hypertension, especially if VUR is present (Hodson
& Edwards 1960, Ransley et al. 1984, Risdon et al. 1994). The causal relationship
between childhood UTIs and their severe long-term consequences and the
rationale involved in evaluating their significance have nevertheless been
questioned (Chambers 1997, Stark 1997, Round et al. 2012).
2.3.1 Chronic kidney disease
Childhood UTIs are found to cause kidney scarring, especially when VUR is
present, and it is claimed that they may lead to impaired kidney function
(Jakobsson et al. 1999b, Wennerstrom et al. 2000, Lahdes-Vasama et al. 2006,
Swerkersson et al. 2007). The presence of scars in the first intravenous urography
(IVU) after the first UTI is more common in boys than in girls (Wennerstrom et al. 2000), but the majority of the scars detected in the acute or early phase disappear
within two years (Jakobsson et al. 1994). Less than 40% of the children have
permanent scars in the kidneys after the first episode of pyelonephritis, and it is
rare for scars to be found at new sites in follow-up DMSA scans (McKerrow et al. 1984, Jakobsson et al. 1994, Vernon et al. 1997, Smellie et al. 1998, Hoberman et al. 1999, Ilyas et al. 2002, Shaikh et al. 2010). The most severe consequence of
childhood UTI has been thought to be CKD, but evidence for an association
between childhood UTIs, renal damage and CKD in children with structurally
31
normal kidneys is scarce (Hellerstein 2000, Round et al. 2012). The aetiological
role of childhood UTIs as a cause of CKD is not well known, and there are only a
few follow-up reports available on patients who had had UTIs decades earlier
(Hellerstein 2000). The active treatment of children with VUR has not reduced
the incidence of end-stage renal disease caused by reflux nephropathy (Craig et al. 2000).
2.3.2 Other consequences
Diastolic blood pressure in adulthood is higher in patients who had had febrile
UTIs and kidney scars in childhood than in controls or patients without scars, but
the scars do not affect systolic blood pressure and hypertension is rare (Jacobson
et al. 1989, Martinell et al. 1990, Martinell et al. 1996, Lahdes-Vasama et al. 2006). Thus UTIs in childhood are not a significant risk factor for hypertension
(Wennerström et al. 2000a). Women with scarred kidneys after UTIs or ABU in
childhood seem to have more pregnancy complications such as arterial
hypertension and pre-eclampsia than those without such a history (Sacks et al. 1987, Jacobson et al. 1989, McGladdery et al. 1992, Smellie et al. 1998).
2.4 Prevention of urinary tract infections in childhood
The objectives of the prevention of UTI are to reduce the burden of the related
symptoms and to protect the kidneys from damage caused by recurrent episodes
of pyelonephritis.
2.4.1 Antimicrobials
Antimicrobials are effective to a limited degree in preventing recurrences of UTI
in children (Uhari et al. 1996b, Roussey-Kesler et al. 2008, Craig et al. 2009,
Williams & Craig 2009). Craig et al. (2009) have reported recently that
trimethoprim-sulphamethoxazole reduced the absolute risk of UTI by 6
percentage points (13% vs. 19%), that the number it was necessary to treat to
eliminate one case was 14, and that 67% of the infections in the antibiotic group
were caused by resistant bacteria, the use of prophylactic antibiotics having
increased the risk of infections caused by these. Likewise, there are reports of
antimicrobials having no effect on the recurrence of UTI but increasing the risk of
infections caused by resistant bacteria (Garin et al. 2006, Conway et al. 2007,
32
Montini et al. 2008). Differences in compliance between the antibiotic and
placebo groups do not explain the poor efficacy revealed by these studies.
Furthermore, prophylactic antibiotics are not effective in preventing UTIs or the
progression of renal scars in patients with VUR (Garin et al. 2006, Montini et al. 2008, Pennesi et al. 2008, Craig et al. 2009). In a small series (N=18) studied by
Lohr et al. (1977) nitrofurantoin was more effective than a placebo for preventing
UTIs (0 vs. 1.7 episodes/PYR) and bacteruria (0.2 vs. 2.5 episodes/PYR) in girls
with a history of recurrent UTIs (Lohr et al. 1977). Similarly, nitrofurantoin is
more effective in preventing recurrences of UTI in children than are
sulphonamides (9.4 vs. 27 episodes/100 person years at risk) (Uhari et al. 1996b).
2.4.2 Dietary factors
Many plants produce antimicrobial compounds in response to microbial invasion,
and when ingested, these compounds may modulate the bacterial flora in the gut.
Changes in cattle nutrition have been shown to result in changes in their colonic
bacterial flora (Callaway et al. 2003). Berries of the Vaccinium family (bilberries,
lingonberries and cranberries) and their extracts have marked activity against
many human bacteria in vitro (Ofek et al. 1991, Howell et al. 1998, Burger et al. 2000, Ho et al. 2001, Puupponen-Pimiä et al. 2001, Reid et al. 2001, Cavanagh et al. 2003, Nogueira et al. 2003, Weiss et al. 2004). The ingestion of fermented
milk products containing probiotics has also been said to reduce the risk of UTI
(Kontiokari et al. 2003).
2.4.3 Cranberry
Cranberries, like other berries of the Vaccinium family, have a wide antimicrobial
spectrum in vitro (Table 1). Cranberry products appear to be able to prevent UTI
recurrences and bacteruria in adults, but evidence for their efficacy in children is
lacking (Blatherwick 1914, Avorn et al. 1994, Schlager et al. 1999, Kontiokari et al. 2001, Stothers 2002, Jepson et al. 2004, Wing et al. 2008). Three randomized
controlled trials (RCT) have shown that cranberry juice can prevent bacteruria in
elderly people and UTIs in women, and it has been suggested as an alternative to
antimicrobials for UTI prevention (Avorn et al. 1994, Kontiokari et al. 2001,
Stothers 2002). In a recent RCT, however, cranberry juice was not effective in
reducing the number of UTI recurrences among college women (Barbosa-Cesnik
et al. 2011).
33
The mechanism of action of cranberry juice is uncertain. It does not reduce
the pH of the urine significantly nor does it produce enough hippuric acid to
explain its antibacterial activity (Avorn et al. 1994, Di Martino et al. 2006, Naves
et al. 2008a). It is thought to act by inhibiting the adhesion of E. coli to
uroepithelial cells (Sobota 1984, Howell et al. 1998), since cranberry extract and
its urinary metabolites have been shown to reduce bacterial adherence in urine in
a dose-dependent manner (Di Martino et al. 2006, Gupta et al. 2007, Lavigne et al. 2008, Howell et al. 2010). This effect has been demonstrated in urine a few
hours after the ingestion of cranberry juice and it is not dependent on the
expression of genes encoding type P pili (Howell et al. 2005, Di Martino et al. 2006, Tao et al. 2011).
Cranberries have been shown to contain two compounds that have anti-
adherence activity, fructose and proanthocyanidins (PAC). The inhibition of type
1 fimbria-mediated adherence by cranberries has been attributed to fructose, but
the effect has only been demonstrated in vitro, and other fruits and berries
containing comparable amounts of fructose do not have the same anti-adhesion
activity (Zafriri et al. 1989, Howell et al. 2005). Cranberry PACs with A-type
linkage inhibit the adherence of P-fimbriated strains of E. coli (Zafriri et al. 1989,
Howell et al. 1998, Foo et al. 2000, Howell et al. 2005), and PACs have been
shown in animal models to prevent UPEC from invading the kidney cells
(Tufenkji et al. 2010). Cranberry-derived PACs are also known to inhibit the
flagellum-mediated motility of UPEC in vitro (Hidalgo et al. 2011a). Furthermore,
cranberries and their PACs act as iron chelators and inhibit the growth of UPEC
by inducing a state of iron depletion in bacteria (Hidalgo et al. 2011b, Lin et al. 2011). The drinking of cranberry juice has not been found to lead to any
significant increase in the concentration of PACs in the urine, however (Valentova
et al. 2007).
Cranberries may also act by inhibiting the formation of biofilm by UPEC
(Reid et al. 2001, Di Martino et al. 2005, Valentova et al. 2007, Laplante et al. 2012), but the results regarding the effect of ingested cranberry juice on UPEC
biofilm formation in urine are inconsistent and highly dependent on the methods
used (Reid et al. 2001, Valentova et al. 2007, Naves et al. 2008b, Laplante et al. 2012, Tapiainen et al. 2012).
34
2.4.4 Other preventive measures
Patients are often advised to increase their fluid intake in order to control or
prevent UTIs, but there is no evidence to support such a recommendation (Beetz
2003, Gray & Krissovich 2003). Treating constipation is effective in correcting
voiding dysfunction and preventing UTI recurrences in children (Neumann et al. 1973, Loening-Baucke 1997), while recurrent UTIs in adults can be prevented by
vaginal mucosal immunization with vaccines containing killed uropathogens, the
efficacy of this vaccination being dependent on the HLA-DR phenotype of the
patient, for example (Hopkins et al. 1999a). Circumcision reduces the risk of UTI
in boys and may be cost-effective, but as only 1% of boys with a normal urinary
tract have UTI in childhood, the number of normal boys that it is necessary to
treat to prevent one UTI is more than 100 (To et al. 1998, Schoen et al. 2000,
Singh-Grewal et al. 2005). On the other hand, the number of boys with recurrent
UTIs or significant urinary tract abnormalities that is was necessary to treat per
prevention was 4–10, so that they may benefit from circumcision (Singh-Grewal
et al. 2005). Colonization of the periurethral area with Lactobacilli may reduce
the risk of recurrent UTIs, but oral administration of probiotics is not effective
(Reid et al. 1992, Raz & Stamm 1993, Kontiokari et al. 2001).
Tabl
e 1.
In v
itro
ant
imic
robi
al e
ffec
ts o
f Va
cc
iniu
m b
erry
ext
ract
s on
cer
tain
bac
teria
and
fung
i.
Bac
teria
E
xam
ple
of d
isea
ses
in
hum
ans
Gro
wth
or a
dhes
ion
inhi
bitio
n in
vitr
o by
R
efer
ence
Blu
eber
ryC
ranb
erry
Li
ngon
berr
y
Act
inobaci
llus
act
inom
yces
Den
tal c
arie
s -
- Y
es
Ho
et
al.
2001
Bifi
dobact
erium
lact
is
- N
oN
o N
o P
uupp
onen
-Pim
iä e
t al.
2001
Candid
a a
lbic
ans
Ora
l thr
ush
-N
o -
Cav
anag
h et
al.
2003
Clo
stridiu
m p
erf
rin
gens
Gas
gan
gren
e -
Yes
-
Cav
anag
h et
al.
2003
Esc
herici
a c
oli
UTI
, sep
sis
Yes
Yes
Y
es
Puu
ppon
en-P
imiä
et
al.
2001
, Cav
anag
h et
al.
2003
, Ofe
k e
t a
l. 19
91, H
owel
l et
al.
1998
,
Nog
ueira
et
al.
2003
, Rei
d et
al.
2001
Ente
roco
ccus
fae
calis
P
erito
nitis
N
oY
es/N
o N
o C
avan
agh
et
al.
2003
, Puu
ppon
en-P
imiä
et
al.
2001
Haem
ophilu
s in
fluenza
e
Otit
is, s
inus
itis
-Y
es
- M
cRea
et
al.
2002
Helic
obact
er
pyl
ori
G
astri
tis
-Y
es
- B
urge
r et
al.
2000
Lact
obaci
llus
sp.
- Y
esN
o N
o W
eiss
et
al.
2004
Lis
teria m
ono
cyto
genes
Mis
carr
iage
-
Yes
-
Nog
ueira
et
al.
2003
Myc
obact
erium
ph
lei
- -
Yes
-
Cav
anag
h et
al.
2003
Porp
hyr
om
onas
gin
giv
alis
D
enta
l car
ies
--
Yes
H
o et
al.
2001
Pre
vote
lla in
term
ed
ia
Den
tal c
arie
s -
- Y
es
Ho
et
al.
2001
Pse
udom
onas
aeru
gin
osa
W
ound
infe
ctio
n -
Yes
-
Cav
anag
h et
al.
2003
Salm
onella
sp.
Ent
eriti
s Y
esY
es
Yes
C
avan
agh
et
al.
2003
, Nog
ueira
et
al.
2003
Shig
ella
son
nei
Ent
eriti
s -
Yes
-
Cav
anag
h et
al.
2003
Sta
phyl
oco
ccus
aure
us
Sep
sis,
impe
tigo
-Y
es
- C
avan
agh
et
al.
2003
Str
epto
cocc
us
muta
ns
Den
tal c
arie
s -
Yes
-
Wei
ss e
t al.
2004
- = n
ot te
sted
35
36
37
3 Aims of the research
The specific aims of the research were:
1. To find out whether the formation of an Escherichia coli biofilm is important
for the pathogenesis of UTI, and how it is associated with the level of UTI (I).
2. To determine the aetiological fraction of UTI in childhood as a cause of CKD
(II).
3. To evaluate whether cranberry juice is effective, safe and acceptable for the
prevention of UTIs in children (III and IV).
38
39
4 Biofilm formation by uropathogens as an aspect of pathogenesis (I)
4.1 Subjects
We identified 70 patients with acute symptomatic UTI (34 children and 36 adults)
from whom we had causative E. coli strains available (Table 2). Twenty-one of
these patients had participated in our previous clinical trial to evaluate the use of
cranberry-lingonberry juice for the prevention of UTIs in women (Kontiokari et al. 2001). The diagnosis of UTI was based on clinical signs and/or symptoms and
bacteria growing in a urine culture obtained by suprapubic aspiration or more than
105 CFU/ml of a single pathogen in a urine culture obtained by clean voiding or
transurethral catheterization. If the sample had been taken by the urine pad/bag
technique the criteria for UTI diagnosis were pyuria (> 5 leukocytes/field) and
more than 105 CFU/ml of a single pathogen growing in two consecutive urine
cultures (American Academy of Pediatrics 1999). Growth of 104–105 CFU/ml of a
single pathogen was considered diagnostic if the urine nitrite test was positive.
Pyelonephritis was defined as UTI associated with a fever of over 38.5 ºC and/or
a C-reactive protein value greater than 40 mg/l (Jodal et al. 1975). Patients were
diagnosed as having urosepsis if they had UTI and a blood culture positive for the
same pathogen during the same disease period.
Table 2. Diagnoses, by age and gender of the patients.
Characteristic Cystitis (n=43) Pyelonephritis (n=11) Urosepsis (n=16)
Female/Male 43/0 8/3 11/5
Age, years
Mean
Range
19.8
1.8-57.8
3.6
0.1-13.5
63.7
0.5-97.3
All the pyelonephritis patients were children and all but one of the urosepsis
patients were adults. Twenty-seven of the 34 patients younger than sixteen years
of age had undergone diagnostic imaging of the urinary tract, 20/27 of these (74%)
having no urinary tract abnormalities, 4/27 having grade 1–2 vesicoureteral reflux
(VUR), 2/27 having grade 3–4 VUR and one having a neurogenic bladder. The
frequency of VUR among the child UTI patients in this population (22%) is in
accordance with previous figures (Jacobson et al. 1999). Imaging results were
available for eight adult patients, all of whom had normal findings.
40
4.2 Methods
Bacterial strains and growth conditions
The uropathogenic E. coli strains were frozen and stored in skimmed milk at
−20 °C (strains isolated from recidive infections at −80°C), revived on blood agar
and grown overnight in Luria broth at 35 °C. The susceptibility of these strains to
antimicrobials was determined by the plate diffusion method from urine (all
strains) and blood samples (strains causing urosepsis). A strain was classified as
having antimicrobial resistance if it was resistant to at least one of the antibiotics
tested (Table 3). The strains with intermediate resistance were included in the
susceptible group. This classification into resistant and susceptible strains was
based on the findings in urine samples. One patient was excluded because of
missing susceptibility data. The frequency of antimicrobial resistance is in
accordance with figure published for Finland (Finnish Study Group for
Antimicrobial Resistance 2008).
Table 3. Antibiotics against which the resistance of E. coli strains was tested, and their mean optical density (OD) values by susceptibility group (69 samples)1.
Antimicrobials Susceptible Resistant Intermediate
n OD n OD n OD
Cefuroxime 68 0.51 1 0.37 0 -
Cephalexin 59 0.52 4 0.32 6 0.48
Ciprofloxacin2 36 0.47 0 - 0 -
Mecillinam 66 0.51 1 0.53 2 0.34
Nitrofurantoin 69 0.51 0 - 0 -
Trimethoprim 58 0.54 11 0.35 0 -
Trimethoprim-
sulphamethoxazole3
30 0.57 4 0.36 0 -
1 1 patient´s urine sample was missing, 2 Not tested in paediatric samples (except 1 for urosepsis
sample), 3 tested only in paediatric samples
Verification of biofilm formation
The formation of organized biofilm structures was verified by scanning electron
microscopy (SEM) in 22 selected samples and the viability of the bacteria with
LIVE/DEAD Baclight® staining followed by confocal scanning laser microscopy
(CSLM) in 29 selected samples. Biofilm formation was assessed by subculturing
41
20 µl of an overnight Luria broth in 800 µl of Dulbecco’s modified Eagle’s
medium (0.45% glucose) in each well of a 24-well flat-bottomed polystyrene
microtitre plate containing removable plastic coverslips. Incubation was carried
out anaerobically at 37 °C. For practical reasons the incubation time was 48 h.
After the incubation period the samples were rinsed once with sterile water and
fixed in 4% formaldehyde overnight for SEM. The fixed samples were
dehydrated with 25%, 50%, 75% and 96% ethanol for 20 minutes at room
temperature and finally air-dried. SEM was carried out on a Jeol JSM-6400
microscope in the Department of Electron Optics, University of Oulu. The
samples for CSLM were rinsed once with sterile water after incubation, following
which 2 µl of LIVE/DEAD Baclight® stain was added and they were scanned for
30 minutes or less with a Zeiss LSM 510 microscope at the Department of
Anatomy, University of Oulu.
Optical density as a measure of biofilm formation
The biofilm formation capacity of all the E. coli strains was tested by subculturing
5 µl of an overnight Luria broth in 200 µl of Dulbecco’s modified Eagle’s
medium (0.45% glucose) in each well of a 96-well flat-bottomed polystyrene
microtitre plate. E. coli ATCC 10798 was used as a positive control for biofilm
formation and Luria broth as a negative control (Rediske et al. 1999).
Three wells were used for each sample. The 72 h incubation was carried out
anaerobically at 37 °C. After discarding the medium, the microtitre plate wells
were rinsed once with sterile water, stained with 1% crystal violet for 15 minutes
and rinsed twice with sterile water. The presence of a biofilm was assessed after
eluting the attached crystal violet with 96% ethanol and measuring the optical
density (OD) of the eluted material at 570 nm (Multiscan MCC/340 P). The
results are presented as means of three parallel samples. The mean OD of the
biofilm-positive control sample (5 measurements performed on different cultures,
E. coli ATCC 10798) was 0.72 (range 0.46–1.31) and that of the negative control
sample (3 measurements on different cultures, Luria broth) was 0.22 (range 0.15–
0.29). In order to test the validity of the method, we compared the measured ODs
with the findings in the SEM and CSLM images. The E. coli attached to the
polystyrene microtitre plates were alive and had formed organized biofilm
structures when the OD of the strain was higher than 0.50 (Figure 2).
42
Fig. 2. SEM and confocal CSLM images of biofilm-negative (A and B, optical density 0.48) and biofilm-positive (C and D, optical density 1.27) E. coli strains. Almost all the stained material in the CSLM images (B and D) is green, indicating live bacteria.
Statistical analysis
The differences in optical density between the two susceptibility groups were
tested using the Mann-Whitney U test and those between the three diagnostic
groups using the Kruskal-Wallis one-way analysis of variance. If the Kruskal-
Wallis analysis indicated statistically significant differences, post hoc
comparisons between the pairs were carried out. The differences in the
proportions of biofilm formation between the two susceptibility groups were
tested using the SND test and those between the three diagnostic groups using the
chi-square test. In the case of a statistically significant P-value, post hoc
comparisons of multiple pairs were performed using the SND test according to
our a priori hypothesis (Armitage & Matthews 2002). The statistical analyses
were carried out with SPSS 15.0 for Windows and the StatsDirect statistical
software, version 2.5.6.
43
4.3 Results
The mean OD of all the E. coli strains was 0.50 (range 0.21 to 1.45, SD 0.30).
Taking this figure as the cut-off point for in vitro biofilm formation on the basis of
findings in the SEM images, 22/70 (31%) of the E. coli strains could be said to
have been biofilm-positive. The ODs of the causative strains did not correlate
with the age of the patient (r=-0.12, P=0.32), and the severity of infection was
similarly not dependent on the age of the patient (the mean age of the patients
with invasive infections was 39 years and that of the patients with cystitis 20
years, P=0.32). The mean ODs of the three diagnostic groups differed
significantly (P=0.03). The strains isolated from the patients with pyelonephritis
had significantly higher ODs than those from the patients with cystitis (difference
in the means 0.19, 95% confidence limits (CL) 0.06 to 0.32, P=0.02) (Figure 3),
but the urosepsis patients did not differ significantly in this respect from either the
cystitis patients (difference in the means 0.12, 95% CL -0.06 to 0.13, P=0.68) or
the pyelonephritis patients (difference in the means 0.07, 95% CL -0.04 to 0.32,
P=0.28). Altogether 11/43 (26%) of the E. coli strains isolated from cystitis
patients, 6/11 (55%) from pyelonephritis patients and 5/16 (31%) from urosepsis
patients were positive for biofilm formation. When the children and adults were
tested separately, the difference in the mean OD between the strains causing
invasive infections and those causing cystitis was 0.14 (95% CL -0.02 to 0.31,
P=0.06) in the children and 0.16 (95% CL -0.03 to 0.15, P=0.22) in the adults.
Fig. 3. Biofilm-forming capacity of E. coli strains cultured from patients with different levels of urinary tract infection. The dotted line indicates the cut-off optical density value for biofilm formation.
44
Altogether 8/43 (19%) of the cystitis episodes, 1/11 (9%) of the pyelonephritis
episodes and 6/15 (40%) of the urosepsis episodes were caused by a resistant
E. coli strain as defined in the plate diffusion assay. The E. coli strains collected
from the urine and blood of the urosepsis patients were similar in terms of their
susceptibility. The susceptible strains had significantly higher ODs than the
resistant strains (difference in the means 0.21, 95% CL 0.03 to 0.27, P=0.016),
but there was no difference in age between the patients from whom the
susceptible and resistant strains had been isolated (25.7 vs. 30.7 years, difference
5 years, 95% CL -20.1 to 10.8, P=0.61) (Figure 4). One out of fifteen (7%) of the
resistant E. coli strains and 21/54 (39%) of the susceptible strains formed biofilms
(difference 32%, 95% CL 5% to 48%, P=0.015). The only biofilm-positive
resistant strain had an OD of 0.53.
Fig. 4. Biofilm-forming capacity of antimicrobially resistant and susceptible E. coli strains. The dotted line indicates the cut-off optical density value for biofilm formation.
45
5 Long-term consequences of urinary tract infections in childhood (II)
5.1 Methods
Data sources
We sought evidence of a causal relationship between childhood UTIs and CKD
by performing a systematic literature review and analysing the causes of CKD in
patients who were monitored or treated at Oulu University Hospital or who had
died while undergoing renal replacement therapy (RRT) before enrolment in the
present series. To identify congenital kidney anomalies, we reviewed the first
kidney images systemically for structural abnormalities. VUR was not considered
a structural abnormality.
Literature review
A systematic literature search for the period from January 1966 to August 2009
was conducted with the PubMed database, using the Medical Subject Headings
search terms “kidney failure, chronic,” “renal insufficiency, chronic,” or “uremia”
and “urinary tract infections,” “pyelonephritis,” or “cystitis” (Figure 5). The
searches were limited to publications in English and to human subjects, and the
results were combined. All of the titles and abstracts found were reviewed, and
papers that might only possibly have dealt with the relationship between
childhood UTIs and chronic kidney damage were read and evaluated. Papers were
included if they reported on childhood UTI in patients with CKD or on clinically
significant long-term renal consequences of childhood UTIs. Papers concerning
only patients with congenital malformations were excluded. The reviews and
articles included were searched manually for additional references. The focus of
the review was on the number of childhood UTIs and kidney imaging findings in
patients with CKD without a definitive non-infectious cause. We followed the
PRISMA guidelines and used the PRISMA checklist when conducting and
reporting the literature review (Moher et al. 2009).
46
Local patients
The hospital records of all 366 patients who had been treated or monitored on
account of CKD at Oulu University Hospital (which serves an area with 384 000
inhabitants) in 2005–2006 were reviewed (Figure 6 and Table 4). We estimated
glomerular filtration rates by the Modification of Diet in Renal Disease method
and defined CKD on the basis of an estimated glomerular filtration rate of <40
mL/minute per 1.73 m2 or kidney disease necessitating RRT (haemodialysis,
peritoneal dialysis, or kidney transplantation) for >3 months (Levey et al. 1999).
The 308 patients who had a defined non-infectious cause of CKD (e.g.
glomerulonephritis or diabetic nephropathy) were excluded from the more
thorough investigations (Figure 6). Patients with VUR, nephrosclerosis, or
diseases or malformations affecting only one kidney were included in the review
analysis. The remaining 58 patients were asked for interviews, and 54 (92%) of
them gave their informed consent and completed a structured questionnaire about
childhood UTI. The hospital records of the four patients who declined to be
interviewed were reviewed (Figure 6). The patient records of the 54 patients who
gave their informed consent were obtained from the community health centres
where they had been treated or monitored in childhood. The patients and their
records were identified on the basis of individual social security numbers, which
have been issued to every Finnish citizen since 1964.
47
Fig. 5. Results of the literature search on childhood UTI in patients with chronic kidney disease.
48
Fig. 6. Patients treated or monitored at Oulu University Hospital on account of chronic kidney disease (CKD) and their UTIs in Childhood.
49
Table 4. Patients monitored at Oulu University Hospital on account of CKD.
Characteristic Definitive non-infectious
cause for CKD1
No definitive non-infectious cause for CKD
(n=308) No symptomatic UTIs
in childhood (n=45)
Symptomatic UTI in
childhood (n=13)2
Age, mean (range), y 54.1 (1.4-85.2) 61.2 (35.1-92.6) 54.7 (39.9-71.1)
Born after 1960, no. (%) 93 (30) 17 (38) 4 (31)
Female, no. (%) 124 (40) 19 (42) 11 (85)
Current therapy, no. (%)
Predialysis 57 (19) 7 (16) 2 (15)
Peritoneal dialysis 21 (7) 3 (7) -
Hemodialysis 93 (30) 19 (42) 1 (8)
Renal transplant 137 (44) 16 (36) 10 (77) 1 Childhood UTI history was not reviewed.
2 For more detailed information, see Table 7.
Abbreviations: UTI=Urinary tract infection, CKD=Chronic kidney disease
Registry data
Data on patients undergoing RRT were collected from the Finnish Registry for
Kidney Diseases, which has been estimated to cover 97% to 99% of all Finnish
RRT patients since 1964 (Finnish Registry for Kidney Diseases 2008). Diagnoses
for patients who had died before the beginning of the present work were collected
from this source.
Ethics approval
The protocol for the invstigation was evaluated and approved by the ethics
committee of the Northern Ostrobothnia Hospital District (Oulu, Finland).
5.2 Results
Literature review
Our systematic literature search yielded a total of 781 papers, the titles and
abstracts of which led to the selection of 39 for further evaluation (Figure 5).
Finally, we found 10 papers reporting results regarding childhood UTIs for 1576
patients with CKD or other clinically significant long-term consequences of
50
childhood UTI (Table 5). None of the studies had been designed to evaluate the
aetiological fraction of UTI in childhood as a cause of CKD, and none of the
reports enabled population-based occurrence figures be calculated. The patient
series were in all cases based on tertiary hospitals serving large populations.
Four of the papers described prospective follow-ups of patients with UTI and
the risk of complications (e.g. scars, VUR) in childhood (Table 5) (Jacobson et al. 1989, Martinell et al. 1996, Smellie et al. 1998, Wennerström et al. 2000b).
Patients with obstructive malformations were excluded. These four papers
included a total of 435 patients, whose ages at the end of the follow-up periods
were between 7 and 52 years. Five of these patients (patients A–E) developed
CKD before the end of the follow-up period (Table 6), all five having had severe
bilateral scars at their first kidney examination and 3 having already had impaired
renal function in childhood. In addition, 1 girl with severe bilateral scars and
impaired renal function in childhood had died as a result of cerebral haemorrhage
attributable to uncontrolled hypertension at the age of 25 years (not included in
Tables 5 and 6) (Smellie et al. 1998).
The remaining six papers examined the causes of CKD for a total of 1141
consecutive patients referred to the renal units of hospitals (Table 5) (Schechter et al. 1971, Murray & Goldberg 1975, Stewart et al. 1975, Huland et al. 1979,
Esbjörner et al. 1997, Sreenarasimhaiah & Hellerstein 1998). Of these patients,
934 had a definitive non-infectious cause of CKD, whereas only 17 out of the
remaining 207 were reported to have experienced symptomatic UTIs at any time.
Seven of the 17 patients (patients F–L) had experienced symptomatic UTIs in
childhood (Table 6), four of them having exhibited structural abnormalities in
their first kidney images. The remaining 3 patients had chronic pyelonephritis
diagnosed in adulthood, by means of a biopsy in 2 cases. The structures of their
kidneys were not evaluated or reported in childhood. In conclusion, only 3 of the
1576 patients identified in the literature search (patients J–L) had childhood UTIs
as a possible cause of CKD, and unfortunately there were no data available on the
structure of their kidneys before the recurrences of UTI.
Local patients
In our review of the data for patients with CKD treated at Oulu University
Hospital, 13 of the 58 patients without a specific non-infectious cause of CKD
had a history of symptomatic UTIs in childhood (Table 7). They all had structural
abnormalities found in their first kidney imaging examination, of which had
51
mostly been performed in childhood (5 patients) or early adulthood (4 patients).
VUR (which was not considered a structural abnormality) had been diagnosed in
3 patients. Six of the 13 patients exhibited urethral obstructions or congenital
anomalies severe enough to cause kidney failure without other contributing
factors, and four of the remaining 7 patients demonstrated dysplastic or
hypoplastic kidneys in their first kidney images and had experienced only 1 or 2
UTIs during childhood but recurrent UTIs in adulthood. Recurrent UTIs during
childhood may have contributed significantly to the later development of CKD in
3 patients (patients 8, 10, and 11) whose kidney structures had been abnormal
when first examined, in such a way that the abnormalities could have been
observed by ultrasonography.
Among these 3 patients was a 46-year-old woman (patient 8) who had
experienced recurrent febrile UTIs during childhood and recurrent UTIs that
required antimicrobial prophylaxis in early adulthood. Her left kidney was
hypoplastic and the right kidney was seen to be scarred in her first kidney
examination, at the age of 20 years. She received a renal transplant at the age of
39 years. The second patient was a 52-year-old woman (patient 10) who had
experienced 3 UTIs in childhood. She had exhibited a hypoplastic left kidney and
hydronephrosis and deformed calices in the right kidney in her first urographic
study, at the age of 11 years. She had also suffered from hypertension since the
age of 11 years, and her left kidney had been removed because of uncontrolled
hypertension when she was 13 years of age. She had received a renal transplant at
the age of 44 years. The third patient was a 60-year-old woman (patient 11) who
had experienced recurrent UTIs in childhood and had exhibited a shrunken kidney
in her first imaging examination, at the age of 30 years. She had received a renal
transplant at the age of 55 years. If we regard childhood UTIs as a factor
contributing to CKD in this third patient, then the aetiological fraction of
recurrent childhood UTIs as a cause of CKD would be at most 1 in 366 (0.3%).
Tabl
e 5.
Pap
ers
on p
atie
nts
with
UTI
s in
chi
ldho
od a
nd la
ter
CK
D
Stu
dy
Pop
ulat
ion
Age
at e
nd o
f fol
low
-up
perio
d P
atie
nts
with
sym
ptom
atic
UTI
s in
chi
ldho
od a
nd
CK
D w
ithou
t any
def
initi
ve n
on-in
fect
ious
cau
se1
Pro
spec
tive
stud
ies
Sm
ellie
et al.
1998
22
6 pa
tient
s w
ith U
TI a
nd n
on-
obst
ruct
ive
VU
R in
chi
ldho
od
26-5
2 ye
ars
2 gi
rls
Mar
tinel
l et al.
1996
11
1 gi
rls w
ith U
TI o
r AB
U a
nd ri
sks
in
child
hood
16-3
3 ye
ars
Non
e
Wen
ners
tröm
et
al.
2000
68 p
atie
nts
with
UTI
and
non
-obs
truct
ive
scar
s in
chi
ldho
od
7-34
yea
rs
Non
e
Jaco
bson
et
al.
1989
30
pat
ient
s w
ith U
TI a
nd n
on-o
bstru
ctiv
e
scar
s in
chi
ldho
od
22-4
1 ye
ars
1 m
an a
nd 2
wom
en
Ret
rosp
ectiv
e st
udie
s on
patie
nts
with
CK
D
Esb
jörn
er 1
997
All
patie
nts
with
CK
D in
chi
ldho
od in
Sw
eden
0-16
yea
rs
Non
e
Sre
enar
asim
haia
h et
al.
1998
102
child
ren
0-20
yea
rs
1 gi
rl
Ste
war
t et a
l 197
5 40
3 co
nsec
utiv
e pa
tient
s 15
-55
year
s N
one
(tim
ing
of U
TI in
j 3 p
atie
nts
with
unc
erta
in
diag
nosi
s w
as n
ot re
porte
d)
Mur
ray
et al.
1975
32
0 pa
tient
s 10
-81
year
s N
one
(tim
ing
of U
TI in
1 p
atie
nt w
ith
neph
rosc
lero
sis
was
not
repo
rted)
Sch
echt
er e
t al.
1971
17
3 co
nsec
utiv
e pa
tient
s 9-
61 y
ears
2
wom
en
Hul
and
et
al.
1979
25
con
secu
tive
patie
nts
(all
had
VU
R in
adul
thoo
d)
7-61
yea
rs
4 w
omen
Tota
l N
1576
12
1 For
mor
e de
taile
d in
form
atio
n, s
ee T
able
6
52
Tabl
e 6.
Tw
elve
pat
ient
s w
ith C
KD
of
no d
efin
itive
non
-infe
ctio
us c
ause
iden
tifie
d in
the
lite
ratu
re r
evie
w a
s ha
ving
had
UTI
s in
ch
ildho
od.
Pat
ient
The
first
kid
ney
imag
ing
C
linic
al d
ata
Des
igna
tion
Gen
der
Ref
eren
ce
R
enal
par
ench
ymal
find
ings
Ti
min
g
A
Fem
ale
Sm
ellie
et al.
1998
Sev
ere
bila
tera
l sca
rs
7-12
y
H
yper
tens
ion
and
CK
D in
chi
ldho
od, R
Tx w
hen
24 y
B
Fem
ale
Sm
ellie
et
al.
1998
Sev
ere
bila
tera
l sca
rs
7-12
y
H
yper
tens
ion
and
CK
D in
chi
ldho
od, R
Tx w
hen
25 y
C
Mal
e Ja
cobs
on e
t al.
1989
and
1992
S
ever
e bi
late
ral s
cars
1-
13 y
CK
D w
hen
17 y
D
Fem
ale
Jaco
bson
et
al.
1989
and
1992
S
ever
e bi
late
ral s
cars
1-
13 y
CK
D w
hen
31 y
E
Fem
ale
Jaco
bson
et
al.
1989
and
1992
S
ever
e bi
late
ral s
cars
1-
13 y
CK
D w
hen
34 y
F Fe
mal
e S
reen
aras
imha
iah
et al.
1998
B
ilate
ral s
cars
7
y
Rec
urre
nt U
TIs
in c
hild
hood
and
adu
lthoo
d, C
KD
whe
n
22, f
ibro
sis
and
foca
l atro
phy
in b
iops
y w
hen
8 y
G
Fem
ale
Sch
echt
er e
t al.
1971
Roe
ntgo
enog
raph
ic c
hang
es
char
acte
ristic
of C
P
NR
Rec
urre
nt U
TIs
in c
hild
hood
, CP
in a
dulth
ood,
CK
D w
hen
17 y
H
Fem
ale
Sch
echt
er e
t al.
1971
Roe
ntgo
enog
raph
ic c
hang
es
char
acte
ristic
of C
P
NR
Rec
urre
nt U
TIs
betw
een
10-1
2 y,
CP
in a
dulth
ood,
CK
D
whe
n 45
y
I Fe
mal
e H
ulan
d e
t a
l. 19
79
D
yspl
astic
righ
t kid
ney
NR
1
Rec
urre
nt U
TIs
in c
hild
hood
and
adu
lthoo
d, n
ephr
ecto
my
whe
n 35
y, C
P in
adu
lthoo
d
J Fe
mal
e H
ulan
d e
t a
l. 19
79
N
R
27 y
Rec
urre
nt U
TIs
befo
re th
e ag
e of
13,
CP
in a
dulth
ood
(bio
psy)
K
Fem
ale
Hul
and
et
al.
1979
NR
45
y
R
ecur
rent
UTI
s in
chi
ldho
od, C
P in
adu
lthoo
d
L Fe
mal
e H
ulan
d e
t a
l. 19
79
N
R
34 y
Rec
urre
nt U
TIs
in c
hild
hood
, CP
in a
dulth
ood
(bio
psy)
CP
indi
cate
s ch
roni
c py
elon
ephr
itis;
NR
= n
ot re
porte
d; 1 V
UR
dia
gnos
ed a
t the
age
of 4
8 y
53
Tabl
e 7.
Thi
rtee
n pa
tient
s in
Oul
u w
ith C
KD
of n
o de
finiti
ve n
on-in
fect
ious
cau
se w
ho h
ad U
TI in
chi
ldho
od.
Pat
ient
Firs
t kid
ney
imag
ing
No.
of s
ympt
omat
ic U
TIs
C
linic
al d
ata
Hyp
opla
siaa
O
ther
feat
ures
A
ge, y
Chi
ldho
od
Adu
lthoo
d
Des
igna
tion
Gen
der
Age
, y
R
ight
Le
ft
Rig
ht
Left
Obs
truct
ive
urop
athy
1 Fe
mal
e40
No
No
U
reth
ral s
teno
sis
Ure
thra
l
sten
osis
6
Rec
urre
nt
Rec
urre
nt
U
reth
ra d
ilate
d at
6 y
,
rena
l tra
nspl
ant a
t 22
y,
FSG
S in
bio
psy
2 M
ale
52
Yes
Ure
thra
l val
ve
Ure
thra
l val
ve
16
1
0
Ren
al tr
ansp
lant
at 1
9 y
3 M
ale
51
N
o Y
es
U
reth
ral v
alve
U
reth
ral v
alve
1,
5
A fe
w
2
Ren
al tr
ansp
lant
at 4
7 y
Con
geni
tal a
nom
aly
4 Fe
mal
e 41
No
Not
pres
ent
S
cars
A
plas
ia
37
R
ecur
rent
0
H
aem
odia
lysi
s at
41
y,
mul
tiple
mal
form
atio
ns
5 Fe
mal
e 61
Not
pres
ent
NR
Apl
asia
E
ctop
ic
19
A
few
R
ecur
rent
Ren
al tr
ansp
lant
at
35 y
, gyn
ecol
ogic
al
abno
rmal
ities
6 Fe
mal
e 76
Not
pres
ent
Yes
Apl
asia
H
orse
shoe
61
1 A
few
Pre
dial
ysis
at 7
3 y
Hyp
opla
stic
/dys
plas
tic k
idne
ys
7 Fe
mal
e 52
Yes
Y
es
D
ilate
d ur
eter
N
one
12
1
Rec
urre
nt
R
enal
tran
spla
nt a
t 34
y
8b Fe
mal
e 46
Yes
Y
es
S
cars
S
cars
20
Rec
urre
nt
A fe
w
R
enal
tran
spla
nt a
t 39
y
9 Fe
mal
e 60
Yes
Y
es
N
one
Non
e 23
2 R
ecur
rent
Ren
al tr
ansp
lant
at 4
4 y
54
Pat
ient
Firs
t kid
ney
imag
ing
No.
of s
ympt
omat
ic U
TIs
C
linic
al d
ata
Hyp
opla
siaa
O
ther
feat
ures
A
ge, y
Chi
ldho
od
Adu
lthoo
d
Des
igna
tion
Gen
der
Age
, y
R
ight
Le
ft
Rig
ht
Left
10b
Fem
ale
50
Y
es
Yes
Hyd
rone
phro
sis
Non
e 11
Rec
urre
nt
Rec
urre
nt
R
enal
tran
spla
nt a
t
44 y
, nep
hrec
tom
y at
13 y
11b
Fem
ale
44
Y
es
Yes
Non
e N
one
30
R
ecur
rent
N
R
R
enal
tran
spla
nt a
t 55
y
12
Fem
ale
69
Y
es
Yes
Non
e N
one
20
1
Rec
urre
nt
P
redi
alys
is in
her
50s
13
Fem
ale
71
Y
es
Yes
Non
e N
one
39
1
5
Ren
al tr
ansp
lant
at 6
6 y
FSG
S in
dica
tes
foca
l seg
men
tal g
lom
erul
oscl
eros
is; N
R, n
ot re
porte
d.
a Incl
udes
dys
plas
ia.
b Rec
urre
nt c
hild
hood
UTI
s po
ssib
ly c
ontri
bute
d si
gnifi
cant
ly to
the
deve
lopm
ent o
f CK
D.
55
56
Registry data
According to the Finnish Registry for Kidney Diseases, nineteen RRT patients
born in the Oulu University Hospital area after 1960 had died before the
beginning of the present work (Table 8) (Finnish Registry for Kidney Diseases
2008), 16 of whom had a specific non-infectious cause of CKD, while of the
remaining three patients (all male), one with probable VUR during childhood had
died of alcohol-related liver cirrhosis at the age of 39, one with CKD had died of
ileus due to a complication of sepsis at the age of 40 and one with congenital
kidney malformation and obstruction in the urinary tract had refused dialysis and
died of terminal uraemia at the age of 36. None of these three patients had had
UTIs during childhood.
Table 8. Patients monitored in the Northern Ostrobothnia Hospital District who had been born after 1960 and had died before the commencement of the present work.
Renal diagnosis N
Diabetic nephropathy 8
Rheumatoid disease 6
Reflux nephropathy 2
Glomerulonephritis 2
Unspecified uraemia 1
Total N 19
57
6 Cranberry juice for the prevention of urinary tract infections (III and IV)
6.1 Subjects
A group of 263 children aged 1–16 years who had been referred to the paediatric
department of one of four university hospitals in Finland (Oulu, Tampere, Kuopio
and Helsinki) or one of the three central hospitals (Joensuu, Lahti, and Kemi) on
account of verified UTI in the previous two months were recruited for a
prevention study (III) to be carried out over the period 2001–2008. Urinary tract
imaging was performed after the first UTI in the manner recommended in Finland
at the time, that is using urogenital ultrasonography and voiding cystography in
all cases aged <2 years and ultrasonography alone on older children initially,
followed by voiding cystography if the ultrasonography findings were abnormal.
Children with severe genitourethral abnormalities and children requiring
antimicrobial prophylaxis for any reason were excluded (Figure 7).
For the colonization study we recruited a total of 341 children attending day-
care centres in the city of Oulu. Children receiving continuous antimicrobial
treatment or having marked immunological defects were excluded. After signed,
informed consent had been obtained, the children were screened by means of
tympanometry and/or pneumatic otoscopy and only those with normal ear status
were accepted (Figure 8).
58
Fig. 7. Design of the prevention trial (III).
Eligible for the study(gave informed consent)
(n=263)
Cranberry juicefor 6 months(n=129)
Placebo juicefor 6 months(n=134)
Diary for 12 months, control visits or callsevery three months, urine sample wheneverUTI occurred and at 12 months
Comparison
* They contributed days at risk until they dropped out.
16 patients dropped out*7 did not accept the juice9 other reasons
11 patients dropped out*6 did not accept the juice5 other reasons
3 protocol violations1 did not start drinking the juice1 needed antimicrobial prophylaxis1 with VUR gr. III-IV
5 protocol violations5 did not start drinking the juice
Cranberry(n=126)
Placebo(n=129)
Randomallocation
* They contributed days at risk until they dropped out.
59
Fig. 8. Design of the colonization trial (IV).
Recruitment at day-care centers(gave informed consent)
(n=341)
Cranberry juicefor 3 months(n=171)
Placebo juicefor 3 months(n=170)
Diary for 3 months, family physician consultedif symptoms of infection occurred, secondfaecal and nasopharyngeal sample at 3 months.
Comparison
20 patients dropped out*18 did not accept the juice2 lost from follow-up
11 patients dropped out*11 did not accept the juice
4 protocol violations4 did not start drinking the juice
2 protocol violations2 did not start drinking the juice
Cranberry(n=147)
Placebo(n=157)
Randomallocation
Delivery of juices and follow up sheets, firstfaecal and nasopharyngeal sample
* They contributed days at risk until they dropped out. * They contributed days at risk until they dropped out.
60
6.2 Methods
Definition of UTI in the prevention trial
UTI was defined on entry as fever and/or local urinary tract symptoms (dysuria,
abdominal/back pain, strong-smelling urine, enuresis) and growth of a single
bacterial strain of 105 or more CFU/ml in a midstream urine sample or a sample
taken with a urine bag or catheter, or any growth in a suprapubic bladder aspirate
sample. The same criteria for UTI were used during the trial except that two
consecutive midstream or bag samples with the same bacterial growth were
required. The UTI symptoms were defined in the information sheet and a study
diary was given to the parents, who were asked to show it to the attendant
physicians. An interval of ten days or more was required for two consecutive
UTIs to be recorded as separate events. Significant bacteruria was defined as
growth of a single bacterial strain of 105 CFU/mL in a midstream urine sample or
a sample taken with a urine bag.
Randomization
On receipt of a signed declaration of informed consent from the parents, the
children were randomized with a block size of four (prevention study) or six
(colonization study) to receive either cranberry juice or a placebo juice 5 ml/kg up
to 300 ml a day in one or two daily doses for six months (prevention study) or in
three daily doses for three months (colonization study) (Figures 7 and 8). The
code was kept sealed until all the data had been collected and processed. Each
centre (prevention study) received sealed envelopes for randomization purposes
before the beginning of the recruitment phase, so that the clinicians were unaware
of the allocation of individual patients.
Study design
At the first visit the parents completed a questionnaire providing background data,
nutritional status and a medical history and were requested not to give the
children any other Vaccinium products during the period of the trial.
In the prevention study the children were followed up for one year, during
which their consumption of the juice and daily symptoms compatible with UTI
61
were recorded in a diary kept by the parents. This one-year follow-up period was
chosen on the basis of our earlier experience in a trial among adult women
(Kontiokari et al. 2001). The parents were advised to take the child to their own
family physician whenever a fever or local symptoms suggestive of UTI occurred.
When UTI was diagnosed, it was treated with a standard antimicrobial regimen,
while the child continued taking the juice. If the child had three or more UTI
recurrences during the trial, antimicrobial prophylaxis for six months was
recommended. The physicians were asked to record their diagnosis and the
resulting treatment on a follow-up sheet, which was returned to the centre
concerned at the last visit. The results of all urine tests carried out during the
follow-up were collected from the laboratory databases. Compliance with the
protocol was determined on the basis of the self-report sheets and by counting the
empty juice cartridges returned by the parents at the end of the trial.
In the colonization study the parents were advised to take the child to their
own family physician every time he/she had any symptoms of infection such as
fever, cough, rhinitis, earache, sore throat, vomiting, diarrhoea, dysuria, night
restlessness, irritability, rash, poor appetite or conjunctival symptoms. The
physicians were asked to record their diagnosis and the resulting treatment on a
follow-up sheet. When any bacterial infection was diagnosed it was treated with
the standard antimicrobial regimen and the child continued taking the juice.
Symptoms, diagnoses, treatments and doses of the products were recorded on a
self-report sheet by the parents and attending physicians and this sheet was
returned at the last visit.
Products and controls
The commercially available cranberry juice used (Cranberry Classic) contained
41 g of cranberry concentrate, including flavouring, in one litre of juice, while the
placebo drink was almost identical in appearance, smell, taste and colour but did
not contain either fruit or berry extracts. The juices were supplied in similar white,
coded cartridges containing 200 ml. The nutritional values of the drinks
resembled those of commonly available fruit and berry juices. Both products were
manufactured and provided by Ocean Spray Cranberries (Lakeville, MA, USA).
62
Sampling and bacterial identification
Stool samples for the colonization study were taken into dry vials at the start and
at three months, and stored at −70 °C until analysed. Before gas liquid
chromatography (GLC), the samples were processed to separate the bacteria from
other faecal material, as described previously (Moss & Nunez-Montiel 1982,
Eerola & Lehtonen 1988). A GLC profile represents all the bacterial cellular fatty
acids in the sample and thus reflects its microflora and can be used to detect
changes, differences or similarities in bacterial flora between individual samples
or sample groups (Peltonen et al. 1992). The fibrous material from the diet and
eukaryotic cells from the gut wall were removed and the methylated fatty acids
were extracted from the remaining sample. GLC was performed using an HP
6890 gas chromatograph with an Ultra 2 (cross linked 5% PH ME Silicone) 25m
x 0.2mm column (HP 19091B) combined with HP ChemStation analysis software.
In the colonization study nasopharyngeal samples for bacterial culture were
taken at the start and at three months using a calcium alginate swab, which was
immediately transferred to a transport medium (ST-GG tube, National Public
Health Institute, Helsinki, Finland) (Kaijalainen et al. 2004). The samples were
stored at −70 °C until analysed. The four most common respiratory pathogens, i.e.
Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pyogenes, were identified at the National Public Health Institute,
Oulu, Finland, by routine methods that involved comparing their colony
morphology and substrate utilization on plate cultures.
Sample size
A 50% reduction in recurrences was considered to be clinically important in the
prevention study, being an effect that was regarded as attainable on the basis of
our earlier trial (Kontiokari et al. 2001). The sample size calculations were based
on earlier reports in which up to 30% of children were said to experience a new
episode of UTI within twelve months (Mangiarotti et al. 2000, Nuutinen & Uhari
2001). To detect a reduction in recurrences to 15% with a two-tailed α of 0.05 and
a power of 80%, and estimating that 10% of the subjects will drop out, it was
decided that 130 children were needed in each group.
The assumptions used for the sample size calculations in the colonization
study were based on differences in nasopharyngeal carriage. We knew the
63
baseline carriage rates of the most common respiratory pathogens to be around
30% in children attending day care centres and assumed that active treatment
would reduce this to 15% (Uhari et al. 1996a). A reduction of this size with a two-
tailed α of 0.05 and a power of 90% would require a sample size of 152 children
in each group. Given an estimated a dropout rate of 10%, a total sample size of
336 was deemed to be needed.
End points
The primary end point in the prevention study was the occurrence of the first UTI
episode during the 12-month follow-up. Secondary end points were the UTI
incidence density and antimicrobial use.
In the colonization study the primary end points for the trial were differences
in the nasopharyngeal carriage of respiratory pathogenic bacteria, faecal fatty acid
composition and compliance, while the secondary end points were the total
numbers of infection episodes in the groups and the duration of their symptoms. A
new episode of infection was diagnosed only after the child had had at least three
asymptomatic days following the previous episode.
Statistical methods
In the prevention study we used the Kaplan–Meier method to analyse the time
elapsing before the first UTI recurrence and the log-rank test to assess the
differences in cumulative survival functions between the groups. The occurrence
of all episodes of UTI was expressed in terms of incidence density, calculated by
summing the number of UTI attacks and the time at risk in each group and then
calculating the rate of UTI episodes per person-year at risk (PYR). Each patient
contributed days at risk until the point of dropping out or receiving antimicrobials
for any reason (if for UTI, at least 10 days were subtracted), or until the follow-up
ended. For the children who received antibiotic prophylaxis the intervention
ceased at that point. The differences in UTI incidence density between the groups
were tested on the assumption that the occurrence of UTI follows a Poisson
distribution. The differences between the groups in the proportion of children
with at least one UTI recurrence, antimicrobial days per year, antimicrobial
prophylaxis and dropouts were tested with the binomial SND (Standardized
Normal Deviate) test. The data were analysed with PASW Statistics for Windows
(version 18.0) and Stats- Direct software (version 2.7.2).
64
In the colonization study the number of children carrying nasopharyngeal
bacteria at each time point was calculated and the proportions of children showing
no change, changing from positive to negative, or vice versa were determined and
compared between the groups. The fatty acid profiles were analysed by
computerized correlation and cluster analysis to calculate mean correlation values
between isolates belonging to the same or different species, and to establish
cluster analysis dendrograms (Peltonen et al. 1997). The cumulative drop-out rate
was calculated and the children were assigned to three dosage groups, >90%,
70−90% or <70% of the recommended doses taken, and the proportions in each
group counted. The numbers of episodes with any symptoms of infection (fever,
cough, rhinitis, earache, sore throat, vomiting, diarrhoea, irritability or
conjunctival symptoms), symptoms suggesting respiratory infection (fever, cough,
rhinitis, earache, sore throat, irritability or conjunctival symptoms) and symptoms
suggesting enteric infection (vomiting, diarrhoea) were counted and the mean
duration of these symptoms was calculated per child and the mean of the means
compared between the groups. Similarly, the diagnoses given by physicians were
counted and grouped as possible bacterial (acute otitis media, pneumonia,
conjunctivitis, sinusitis, bacterial tonsillitis, skin abscess, impetigo, balanitis or
UTI) or viral (laryngitis, pharyngitis, bronchitis, rhinitis, upper respiratory
infection, varicella, viral tonsillitis or viral rash) infections. The incidence
densities of infection episodes and diagnoses were then calculated by summing
the numbers of episodes and times at risk in each group and then calculating the
rate of episodes per person year (365 days) at risk (PYR). Differences, standard
normal deviations and 95% confidence intervals (CI) were calculated between the
proportions and tested with the standard normal deviation test, t-test or Mann
Whitney U -test, depending on the variable.
Ethics
The Ethical Committee of the Medical Faculty of the University of Oulu
evaluated and accepted the protocols of both studies.
65
6.3 Results
Efficacy
The baseline characteristics of the UTI prevention groups were similar (Table 9).
Eight out of the 263 children were omitted from the analysis because of protocol
violation at entry, so that 255 children were included in the final analyses (Figure
7).
Table 9. Baseline characteristics of the subjects in the cranberry juice and placebo juice groups in the prevention study.
Characteristic Cranberry
(n=126)
Placebo
(n=129)
Girls (%) 115 (91) 117 (91)
Age (years) mean (SD), y 3.8 (2.5) 4.5 (2.9)
No. of toddlers (aged 1-3 years) 56 47
Previous UTI morbidity
Number of UTIs, mean (SD) 1.6 (1.3) 1.6 (1.3)
Children with at least two UTIs 38 (30) 38 (30)
Frequency of intake of berry or
fruit juices1
More than 2 times a week 75 (61) 78 (62)
1-2 times a week 16 (13) 21 (17)
Less than once a week 32 (26) 26 (21)
Frequency of intake of berries2
More than 2 times a week 16 (13) 29 (23)
1-2 times a week 51 (43) 45 (36)
Less than once a week 52 (44) 50 (40)
Constipation3 2 (2) 8 (6)
Vesicoureteral reflux grade I-II 1 (0.8) 1 (0.8)
Duplex system 0 1 (0.8)
All data are no. (%) unless otherwise specified.
Abbreviations: SD, standard deviation; UTI, urinary tract infection. 1 Missing data: 3 children in the cranberry group and 6 children in the placebo group, 2 Missing data: 7 children in the cranberry group and 5 children in the placebo group, 3 Less than 3 bowel movements a week
A total of 27 (11%) children dropped out: 16 (13%) in the cranberry group and 11
(9%) in the placebo group (difference 4%, 95% CI −4% to 12%, P=0.24) (Table
66
10). The most common reason was the child’s reluctance to drink the juice (7 vs.
6 children in the cranberry and placebo groups, respectively).
There were 20 (16%) children in the cranberry group and 28 (22%) in the
placebo group who had at least one recurrent UTI during the 12-month follow-up
(difference −6%, 95% CI −16 to 4%, P=0.21) (Table 10). There was no significant
difference in the timing of the first UTI recurrence (P=0.32) (Figure 9). A total of
74 episodes of UTI were recorded (Table 10). The UTI incidence density per PYR
was significantly lower in the cranberry group, but the proportion of children
having more than one recurrence was similar in both groups (Figure 10, Table 10).
The difference between the groups became apparent 5–6 months after starting the
juice (Figure 10).
Table 10. UTI recurrences and compliance of the subjects in the cranberry juice and placebo juice groups.
Characteristic Cranberry
(n=126)
Placebo
(n=129)
Difference 95% CI P-value
Children with recurrent UTI, no. (%) 20 (16) 28 (22) 6% -16% to 4% 0.21
1 episode 16 17
2 episodes 2 4
3 episodes 1 6
4 episodes 1 1
Number of UTI episodes 27 47
Incidence density of UTI per PYR 0.25 0.41 -0.16 -0.31 to -0.01 0.03
Antimicrobial days per year 11.6 17.6 -6 -7 to -5 <0.001
Antimicrobial prophylaxis, no. (%) 4 (3) 7 (5) -2% -8% to 32% 0.38
Compliance
Doses taken 64% 80% -16% -25% to -7% 0.001
>90 % of doses taken, no. (%) 58 (46) 79 (62)
50-90 % of doses taken, no. (%) 21 (17) 27 (21)
<50 % of doses taken, no. (%) 47 (37) 21 (17)
Dropouts
Dropouts, No. (%) 16 (13) 11 (9) 4% -4% to 12% 0.24
Follow-up days, mean (range) 111 (7-259) 122 (26-259)
Abbreviations: CI, confidence interval; PYR, person-year at risk; UTI, urinary tract infection.
67
Fig. 9. Timing of the first UTI recurrence during the 12-month follow-up in children receiving cranberry juice or placebo juice. The difference between the groups was not significant (P=0.32).
68
Fig. 10. UTI recurrences during follow-up (AUC= area under the curve)
The children in the cranberry group had significantly fewer antimicrobial days,
and antimicrobial prophylaxis was started on account of UTI recurrences in 4 of
126 (3%) cases in the cranberry group and 7 of 129 (5%) in the placebo group
(Table 10). The incidence densities of UTI during the cranberry prevention
regimen were 0.36 per PYR in the cranberry group and 0.54 per PYR in the
placebo group (difference -0.18, 95% CI, −0.42 to 0.06, P=0.15), and those
recorded after that were 0.12 per PYR in the cranberry group and 0.26 per PYR in
the placebo group (difference −0.14, 95% CI, −0.31 to 0.02, P=0.09).
Three of the 23 boys (cranberry 2, placebo 1) and 45 of the 232 girls
(cranberry 18, placebo 27) had at least 1 recurrent UTI. The differences in the
proportions between the cranberry and placebo groups were not statistically
significant. There were a total of 3 recurrent UTI episodes among the boys
(cranberry 2, placebo 1) and 71 among the girls (cranberry 25, placebo 46). The
UTI incidence density per PYR among the girls was significantly lower in the
cranberry group (0.25 vs. 0.46 episodes per PYR; difference 20.19, 95% CI 2.34
to 2.03, P=0.02). When the toddlers (aged 1−3 years) and older children were
69
analysed separately, the results were consistent with the whole group results but
the differences between the cranberry group and placebo group were no longer
statistically significant. E. coli was the dominant bacterial pathogen, found in 79% of the isolates in
both groups (P=0.96). All the baseline samples were negative, and only one
control sample had significant bacterial growth at 12 months. This child had no
symptoms, however, and the growth was considered to represent asymptomatic
bacteruria and was not treated.
Table 11. Baseline subject characteristics and compliance.
Characteristic Cranberry (n=171) Placebo (n=170)
No. of girls, no. (%) 87 (51) 82 (48)
Mean age, years (SD) 4.4 (1.6) 4.1 (1.6)
Breast feeding at any time, no. (%) 154 (95) 162 (9)
Mean duration of day care (mo) 25.1 22.2
Previous history of acute otitis media, no. (%) 138 (87) 144 (88)
Smoking parent, no. (%) 57 (35) 46 (28)
Drop-outs, no. (%) 18 (11) 11 (7)
Refused to drink the juice 4 6
Parents tired of the trial 7 3
Rash 0 1
Gastric symptoms 0 1
Illness during the trial 2 1
Reason not known 3 0
Compliance
90% of doses taken, no. (%) 139 (84) 129 (77)
70–90% of doses taken, no. (%) 17 (10) 31 (18)
<70% of doses taken, no. (%) 9 (6) 8 (5)
Percentages are calculated from the number of subjects giving current information.
Effects on normal flora
The baseline characteristics were essentially the same in both groups in the
colonization study (Figure 8, Table 11). Six children did not start taking the juice
at all and two were lost from the trial without any data on their consumption of
the juice. Samples were taken from all the children at the start and from those still
taking the juice at three months who were available for sampling. This gave us a
total of 341 (100%) baseline nasopharyngeal samples and 304 (89%) control
samples after three months, and similarly 189 (55%) and 156 (46%) faecal
70
samples. The mean age of the children in the colonization study was 4.3 years,
ranging from 1 to 7 years, and the background characteristics of the two groups
were similar (Table 11). The number of dropouts during the three months was 18
(11%) in the cranberry group and 11 (7%) in the placebo group (Figure 8, Table
11).
Fig. 11. Changes in the nasopharyngeal carriage of respiratory pathogenic bacteria during 3 months of receiving cranberry or a placebo drink. The number of children carrying nasopharyngeal bacteria at each point was calculated and the proportions showing no change, changing from negative to positive (-/+), or vice versa (+/-) were counted and a P-value calculated for the difference between the groups. All differences were statistically non-significant (P=0.12–0.92).
There were no statistically significant changes in the nasopharyngeal carriage of
respiratory pathogenic bacteria within or between the groups (Figure 11). The
overall carriage and variation in S. pneumoniae was greatest, ranging from 27% to
46%, and the carriage of M. catarrhalis also varied greatly, from 7% to 25%,
71
compared with the stable carriage of H. Influenzae (11–16%) and S. pyogenes
(2%).
The variation in bacterial fatty acid composition was greater within the
groups than between them, i.e. the bacterial flora changed in both groups over
time (P<0.001) but did not differ markedly between the groups (P>0.05).
Table 12. Infectious symptoms.
Symptom Cranberry
(n=165)
Placebo
(n=168)
Difference
(95% CI)
P-value
Any infection symptom1
Mean number of episodes/PYR 12.0 12.6 -0.7 (-2.3 to 0.9) 0.41
Mean duration of symptoms (days) 8.5 9.2 -0.7 (-3.4 to 1.9) 0.52
Symptoms of respiratory infection2
Mean number of episodes/PYR 11.1 11.6 -0.55 (-2.1 to 1.0) 0.48
Mean duration of symptoms (days) 8.7 9.4 -0.7 (-3.4 to 1.9) 0.46
Symptoms of enteric infection3
Mean number of episodes/PYR 1.9 2.3 -0.45 (-1.1 to 0.2) 0.18
Mean duration of symptoms (days) 0.62 0.66 -0.04 (-0.3 to 0.2) 0.37
The number of episodes is given per person-year at risk (PYR). The mean duration of symptoms was
calculated per child and the means of these means were compared. 1Fever, cough, rhinitis, earache, sore throat, vomiting, diarrhoea, irritability, conjunctival symptoms, 2Fever, cough, rhinitis, earache, sore throat, irritability, conjunctival symptoms, 3 Vomiting, diarrhoea.
There were equal numbers of infection episodes in both groups, and the duration
of symptoms was similar (Table 12). Respiratory symptoms accounted for most of
the symptoms (Table 12). The clinical diagnoses reached by the attendent
physicians were also quite similar in the two groups, with acute otitis media,
upper respiratory infection, acute bronchitis and acute conjunctivitis the four most
common diagnoses, accounting for 71% of all the diagnoses (Table 13).
Accordingly, the need for antimicrobials did not differ between the groups, either.
Acceptability
An average of 64% of the doses of cranberry juice and 80% of the doses of the
placebo in the prevention study were actually taken (P=0.001), indicating that
compliance was better among the children receiving the placebo (Table 10). Only
seven children (5%) in the cranberry group and six (4%) in the placebo group did
not accept the juice. In the colonization study 18 children in the cranberry group
72
(11%) and 11 children in the placebo group (6%) stopped taking the juice before
the end of the trial (Figure 8, Table 11). About 80% of children in both groups
took more than 90% of the doses as instructed (Table 11).
Table 13. Incidence densities of clinical diagnoses, given as the mean number of diagnoses per person-year at risk (PYR)
Diagnosis Cranberry
(n=165)
Placebo
(n=168)
Difference
(95% CI)
P-value
Possible bacterial infections1 2.1 1.9 0.2 (-0.5 to 0.8) 0.6
Possible viral infections2 1.1 1.3 0.2 (-0.7 to 0.3) 0.4
Four most common infections3
Acute otitis media 1.5 1.3 0.2 (-0.3 to 0.8) 0.4
Bronchitis 0.2 0.3 0.1 (-0.3 to 0.2) 0.6
Conjunctivitis 0.1 0.4 -0.2 (-0.5 to 0) 0.05
Upper respiratory infection 0.6 0.8 -0.2 (0.6 to 0.1) 0.2 1 Otitis media, pneumonia, conjunctivitis, sinusitis, bacterial tonsillitis, skin abscess, impetigo, balanitis,
UTI, 2 Laryngitis, pharyngitis, bronchitis, rhinitis, upper respiratory infection, varicella, viral exanthema,
viral tonsillitis, 3 Accounting for 71% of all diagnoses.
73
7 Discussion
7.1 Biofilm formation by uropathogens as an aspect of pathogenesis (I)
We found that one third of the E. coli strains isolated from clinical urine samples
formed a biofilm in vitro. The strains from the patients with pyelonephritis were
more capable of forming a biofilm than those isolated from the cystitis patients.
The biofilm-forming E. coli strains were more susceptible to antibiotics than
those that did not form a biofilm. In two previous studies comparing E. coli strains isolated from prostatititis, pyelonephritis and cystitis patients by similar
methods no differences in biofilm-forming capacity were found between the
strains causing cystitis and pyelonephritis, probably because of the use of a
qualitative method for determining biofilm formation (Kanamaru et al. 2006, Soto
et al. 2007). The proportions of biofilm-positive uropathogenic E. coli isolates
and their susceptibilities to antibiotics reported by us are similar to those found by
Soto et al. (Soto et al. 2007).
We validated our method for measuring biofilm by adjusting the quantitative
optical density values to the qualitative biofilm findings in the SEM and CSLM
images (Costerton et al. 1999, Danese et al. 2000b, Donlan & Costerton 2002,
Branda et al. 2005, Kanamaru et al. 2006, Reisner et al. 2006, Rosen et al. 2007,
Soto et al. 2007), and were thus able to present images of well-organized multi-
layer biofilm structures, including the water channels formed by vital bacteria.
Measurement of the optical density of bacteria attached to microtitre plates
appeared to be a simple and reliable method for quantifying the biofilm formation
ability of E. coli. The biofilm formation detected in an in vitro setting may be different from
that occurring in vivo. In addition to the phenotype and genotype of the bacteria,
the biofilm-forming capability of E. coli is also associated with the growth media,
growth surfaces and other environmental factors (Pratt & Kolter 1998, Reisner et al. 2006, Naves et al. 2008b). In addition to this, the storage of the bacterial
isolates, and especially serial cultures, may affect their genotype and alter their
mode of growth (Fux et al. 2005). We were interested in the ability of E. coli strains to form a biofilm in favourable circumstances, however, and used clinical
strains, which had been frozen, stored and revived only once. Our results
demonstrate an association between biofilm formation and clinical presentation
74
with UTI, which indicates that we were able to measure a real biological
phenomenon.
E. coli has been shown to form persistent intracellular biofilm-like reservoirs
in uroepithelial cells (Mulvey et al. 2000, Anderson et al. 2003, Soto et al. 2006,
Rosen et al. 2007). Living bacteria are occasionally released from the biofilm,
and some of these may start to grow in planktonic form (Hancock et al. 2007). If
the released bacteria are virulent enough they may reinvade the uroepithelium and
cause recurrent cystitis, or ascend to the kidneys and cause pyelonephritis (Soto et al. 2006). Uropathogens frequently gain access to the lower urinary tract, but their
persistence in the bladder and further ascent to the kidneys are more restricted
(Nicolle et al. 2005). The ability of E. coli to form a biofilm provides the
uropathogens with more opportunities to invade those parts of the urinary tract
that are more difficult to reach (Soto et al. 2007). In our material most of the E. coli strains isolated from pyelonephritis patients formed a biofilm and all the
strains with a very low biofilm forming ability were isolated from cystitis patients.
Bacteria in biofilms are protected from antimicrobial agents by reduced
penetration and/or active efflux of the antimicrobial drugs from the biofilm
(Costerton et al. 1999, Stewart & Costerton 2001, Smith 2005, Lynch et al. 2007),
and they are therefore not under the same selection pressure as planktonic bacteria
and do not develop resistance by mechanisms that are manifested in the
laboratory tests used to assess the minimal inhibitory concentration of planktonic
bacteria. The lower penetrance of antibiotics into biofilms may lead to suboptimal
antibiotic therapies, which, apart from causing clinical failures, may also promote
the production of more biofilm, which will further reduce the possibilities for
eradicating the bacteria from the urinary tract (Hoffman et al. 2005).
7.2 Long-term consequences of urinary tract infections in childhood (II)
Our literature search failed to identify any papers designed to evaluate the
aetiological fraction of UTI in childhood as a cause of CKD, or any in which
population based occurrence figures could be calculated. Many of the items on
the PRISMA checklist were not applicable (Moher et al. 2009), and taking into
account the variability in the methods used during a period going back 50 years,
we found just ten reports on childhood UTIs in CKD patients or on clinically
significant long-term renal consequences of childhood UTIs. There were no
75
patients among the 1576 cases reviewed in these reports for whom childhood
UTIs were the main cause of subsequent CKD, i.e. all of the patients who had
experienced childhood UTIs and developed CKD either had a specific non-
infectious cause for their CKD or else had structural kidney anomalies in their
first kidney imaging examination, although there was admittedly no information
on the kidney imaging results for three of the patients who had had UTIs in
childhood. Of the 366 living adult patients with CKD who were monitored in our
hospital, only three had experienced recurrent UTIs in childhood that might have
contributed to the development of CKD, and these had had structurally abnormal
kidneys in their first examinations. When population-based occurrence figures
were calculated from our hospital data, the aetiological fraction of childhood
UTIs as a cause of CKD was less than one percent.
We analysed the data on childhood UTIs for the cohort of all patients treated
or monitored on account of CKD in the geographically defined area of northern
Finland. As the patient records for Finnish children have been available since the
1930s (when infant welfare clinics were introduced), and their reliability and
comprehensiveness were improved under the Primary Health Care Act of 1972,
the Finnish health care system provides reasonably reliable clinical data on
childhood UTIs even for patients who were children 50 years ago. The
significance of UTIs in general, and especially in childhood, as a cause of CKD
was nevertheless substantially smaller in our material than could have been
expected on the grounds of previous registry data. There are several possible
explanations for this. In the first place, we were able to get more detailed
information on the aetiology of CKD and the history of UTIs in childhood than
could be derived from registers, and we were thus able to evaluate the
significance of childhood UTIs alone more specifically and take into account the
existence of structural kidney abnormalities etc. On the other hand, advancements
in diagnostic methods and the treatment of UTIs have also had an impact on the
long-term prognosis for UTI in childhood.
As it is thought that recurrent UTIs in childhood, especially together with
VUR, can lead to development of renal scars, hypertension and kidney failure,
imaging is routinely recommended and performed after the first UTI in order to
identify urinary tract abnormalities and VUR (Jacobson et al. 1989, Jakobsson et al. 1994, Smellie et al. 1998, American Academy of Pediatrics 1999, Vallee et al. 1999, Orellana et al. 2004, Swerkersson et al. 2007). The existing concept of
childhood UTIs as a risk factor for kidney failure is nevertheless based on
analyses of selected patient series. According to our results, VUR with UTI
76
without structural abnormalities in the kidneys seems not to cause CKD, and so,
understandably, active treatment of VUR does not reduce the occurrence of CKD
and large prospective follow-up studies have shown renal function to be well
preserved in patients with VUR (Smellie et al. 1998, Craig et al. 2000, Jodal et al. 2006, Venhola et al. 2006). The renal problems associated with primary VUR are
more probably sequelae of congenital abnormalities such as renal dysplasia than
of VUR itself (Risdon et al. 1993). VUR is a self-limiting, age-related
phenomenon, the prevalence of which among otherwise healthy children is
significantly higher than traditional estimates would lead us to believe, and it is
not associated with urinary tract infections (Köllerman & Ludwig 1967, Hannula
et al. 2010).
It is difficult to differentiate between infectious scars and hypoplasia or
dysplasia associated with congenital malformations. A hypoplastic kidney is
defined as a morphologically normal kidney with nephrons reduced in either
number or size, while a dysplastic kidney is one with a disorganized renal
parenchyma resulting from a failure to undergo normal cellular differentiation
during organogenesis. As a dysplastic kidney is often associated with
malformations in other parts of the urinary tract, renal dysplasia is best regarded
as an abnormality of the whole urinary tract (Risdon et al. 1975). Also, because it
is rare for scars to develop at new sites during follow-up and the scarring rate is
independent of the age at which the kidneys are imaged, it seems that the scars are
signs of congenital abnormalities rather than consequences of UTI (McKerrow et al. 1984, Jakobsson et al. 1994, Coulthard et al. 1997, Vernon et al. 1997, Smellie
et al. 1998, Hoberman et al. 1999). As the dysplastic features may be microscopic
and the dysplastic tissue may be located as islets among normal tissue, renal
dysplasia is often impossible to differentiate from renal hypoplasia or infectious
scars by imaging alone (Risdon et al. 1975). Shrunken or hypoplastic kidneys
found in paediatric patients with CKD by imaging are probably a sign of renal
dysplasia rather than a consequence of UTIs (Risdon et al. 1993). In the case of
all three patients in our series with childhood UTIs that could have contributed to
the development of CKD, hypoplastic kidneys had been seen at their first imaging
examination, and, consistent with earlier suggestions, we think that the patients
had most probably had these changes before the recurrent UTIs occurred (Craig et al. 2000).
The significance of childhood UTIs and the need for interventions to reduce
the risk of their rare long-term consequences are both matters that depend on the
77
point of view adopted by the patient, parents, doctor, health care system etc.
(Chambers 1997). There are many widespread practises in medicine that are not
based on evidence but include unpleasant and possibly harmful procedures which
may lead to unnecessary treatment and follow-up. Because 2% to 8% of all
children experience UTIs at some stage and 20% to 30% of those children have
VUR, many children are subjected to repeated radiological imaging and long-
term antibiotic prophylaxis. The mean effective radiation dose in one fluoroscopic
voiding cystoureterography is 0.5 to 1.5 mSv, the organ-specific radiation dose
for the ovaries is 0.5 to 1.5 mGy, and the mean radiation dose for the supporting
adult is 0.14 mGy (Perisinakis et al. 2006, Sulieman et al. 2007). The effective
doses depend on the age and size of the patient and correspond to more than 50
native chest X-ray examinations or up to 6 months of environmental radiation in
Finland (Perisinakis et al. 2006, Kiljunen et al. 2009).
Since all the patients with UTIs in childhood and subsequent CKD
considered in this work had abnormal findings in their first kidney imaging
examination which could have been observed by ultrasonography,
ultrasonography performed after a UTI in childhood seems to be sufficient to
identify patients at risk of developing CKD. It may be that not even ultrasound is
necessary for all children after the first UTI, but more evidence is needed before
recommendations can be given (Hoberman et al. 2003, Zamir et al. 2004, Miron
et al. 2007, Montini et al. 2009). Furthermore, as children with normal kidneys
have no significant risk of CKD, the objective of preventing UTIs should be to
reduce the distress caused by recurrent episodes. The current recommendation to
search actively for VUR and to protect the kidneys from damage caused by UTIs
by means of long-term antibiotics leads to unnecessary radiation exposure,
imposes a burden on patients and families, and implies unnecessary use of the
health care system.
7.3 Cranberry juice for the prevention of urinary tract infections (III and IV)
Regular drinking of cranberry juice reduced the number of UTI recurrences by
43%, but did not significantly reduce the number of children experiencing at least
one recurrence after the initial episode. Among adults, cranberry juice has been
reported to reduce both the number of patients affected and the number of
episodes (Avorn et al. 1994, Kontiokari et al. 2001, Stothers 2002). We had a
lower than expected recurrence rate among our patient, but as we based our
78
sample size calculations on an expected recurrence rate of 30% by reference to
earlier studies, our trial lacked statistical power (Uhari et al. 1996b, Nuutinen &
Uhari 2001). Nevertheless, only 22% of the control children in our trial
experienced at least one recurrence, which was comparable to the rate of 19%
recorded in a recent UTI prevention trial (Craig et al. 2009). Even though the UTI
incidence density was a secondary outcome, our figures indicate a true protective
effect of cranberry juice against UTI in children comparable to that found in
adults. The preventive effect was evident in the second half of the period, a
finding that is in line with the observation in our earlier trial that cranberry-
lingonberry juice maintained its protective effect against UTI for at least six
months after the women ceased to consume it (Kontiokari et al. 2001).
We also found in our trial that the UTI recurrence rate of the children was
lower than that of the adults, and that the recurrences occurred later (Kontiokari et al. 2001). Whereas 30% of the adult women in the control group had had at least
one recurrence at 3 months, this figure was only 10% in the children’s placebo
group, while at 12 months the figures were 39% and 22%, respectively
(Kontiokari et al. 2001). It seems that children develop recurrences of UTI at a
constant rate over time, an observation supported by findings in other studies
(Nuutinen & Uhari 2001, Craig et al. 2009). The slower and lower UTI
recurrence rate in children may be due to differences in UTI risks and
pathogenesis, which are quite well characterized in adults but not so well in
children (Stull & LiPuma 1991, Mårild & Jodal 1998, Nuutinen & Uhari 2001,
Conway et al. 2007, Shaikh et al. 2008, Craig et al. 2009). Uropathogenic
bacteria are able to create intrauroepithelial pods where they rest in biofilm form
and cause recurrences or relapses when circumstances are suitable (Anderson et al. 2003). This has been suggested as an explanation for the numerous recurrences
that occur in adults soon after an initial UTI.
Cranberry products are thought to act against uropathogenic bacteria, mostly
E. coli, by inhibiting their growth and P-pilia-mediated adhesion, and possibly by
reducing their biofilm production (Sobota 1984, Zafriri et al. 1989, Foo et al. 2000, Reid et al. 2001, Howell et al. 2005, Valentova et al. 2007, Lin et al. 2011).
Our concept of its mechanism of action, however, is based on in vitro studies. The
beneficial effects of cranberry may be experienced in the urinary tract, in the gut
or in both. The selection pressure created in the stool by the presence of cranberry
metabolites may induce a shift towards a less uropathogenic bacterial flora, and
hence prolonged protection against UTI while changes in the composition of the
79
gut flora and the virulence of the pathogens present may take place slowly, so that
the efficacy of cranberry juice for the prevention of UTI will not be seen
immediately. On the other hand, starting the juice 1–2 months after a UTI episode
is too late for preventing the causative pathogens from forming a biofilm in the
uroepithelium during the acute infection. The above mechanism would be in
accordance with our finding that cranberry juice does not prevent the first
recurrence but does reduce the number of subsequent episodes. Thus the
preventive effect would be optimal if the juice was started as soon as possible,
preferably at the same time as antimicrobial treatment, which may itself induce
biofilm formation (Hoffman et al. 2005). The current evidence on the effect of
ingested cranberry on biofilm formation by UPEC does not support the latter
hypothesis, however (Reid et al. 2001, Valentova et al. 2007, Laplante et al. 2012,
Tapiainen et al. 2012), so that, since biofilm formation and the expression of the
genes involved are highly dependent on growth conditions, further studies are still
needed (Naves et al. 2008b).
In a recent RCT, cranberry juice had no effect on recurrences of UTI among
college women (Barbosa-Cesnik et al. 2011). The recurrence rate in that series
was about 50% of that reported by Kontiokari et al. (16% vs. 30%) and the
patients had had fewer previous UTIs (3.7 vs. 6) (Kontiokari et al. 2001, Barbosa-
Cesnik et al. 2011). It may be that the difference in the preventive effect of
cranberry juice between the two trials is associated with differences in the diets of
the populations concerned. If the diet includes factors that induce similar
beneficial changes in bacterial flora to cranberry, the effect of a cranberry
intervention will be less evident. Also, Kontiokari et al. used cranberry-
lingonberry juice, and the lingonberry compound may have had an effect on the
recurrences (Kontiokari et al. 2001).
According to the urine samples taken at the start and end of the trial there was
only one child with asymptomatic bacterial growth, so that we believe that our
results were not compromised by the presence of asymptomatic bacteruria cases.
In addition, we excluded those children who would most urgently need
antimicrobial prophylaxis, because we considered it unethical to leave them
without medication. The group included those with severe VUR, who have the
highest risk of UTI recurrences (Nuutinen & Uhari 2001). The majority of
children who suffer from UTIs have no underlying urinary tract pathology and
may benefit from cranberry juice.
Antimicrobial prophylaxis is of limited efficacy in preventing UTIs (Montini
et al. 2008, Roussey-Kesler et al. 2008, Williams & Craig 2009). The most recent
80
large placebo-controlled trial found a modest reduction in the number of children
having at least one recurrence a year, from 19% to 13%, i.e. a decrease of 6
percentage points, or a relative reduction of 65%, implying that 14 children had to
be treated to prevent one case of UTI (Craig et al. 2009). Long-term prophylactic
antimicrobials may induce antimicrobial resistance, and their use may similarly
involve problems of compliance (Uhari et al. 1996b, Craig et al. 2009, Williams
& Craig 2009). The use of cranberry juice for children seems to share some of
these problems, such as limited efficacy and low compliance but even so,
cranberry juice has many advantages over antimicrobials, as it does not induce
antimicrobial resistance (McMurdo et al. 2009). On the contrary, antimicrobial
consumption was reduced by 34%, or 6 days per patient-year, in the cranberry
group.
Most of the bacteria affected by cranberry and other berries of the Vaccinium
family are harmful to humans, while bacteria considered to belong to the normal
beneficial bacterial flora are not affected. Clinically, cranberry juice, capsules
and/or powder have been shown to reduce UTI recurrences and salival counts of
bacteria causing dental caries (Avorn et al. 1994, Kontiokari et al. 2001, Stothers
2002, Weiss et al. 2004). We were unable to see any change in oral bacterial
carriage, even though cranberries have been found to act anti-adhesively on some
of these bacteria in vitro. Our trial similarly showed that drinking cranberry juice
did not markedly affect the bacterial composition of the stools, and gastric
symptoms, which might be related to disturbed faecal bacterial balance, were rare,
occurring in only 1% of the cranberry group during the three months of the trial.
Most bacteria causing human diseases, especially colonic bacteria, arise from
our own bacterial flora, which is in constant contact with ingested food items. It
would be logical to assume that the risk of contracting infectious diseases could
be affected by our diet, but only a few attempts have been made to evaluate this
association (Peltonen et al. 1997). The consumption of berries, fruits and dairy
products containing probiotic bacteria, for example, has been found to be
associated with a decreased risk of UTI (Kontiokari et al. 2003, Kontiokari et al. 2004). In our trial, adding cranberry juice to the normal diet did not alter the
frequency of common infectious diseases in the children over a period of three
months. Most of the infectious episodes were respiratory infections, and there
were no differences in the number of episodes or the duration of the symptoms
with respect to any of the diseases evaluated. Acute otitis media was the most
common bacterial disease and upper respiratory infection the most common viral
81
disease. Some infections such as enteric infections or UTIs were so rare that no
comparisons could be made. The children had an average of three infectious
episodes during the three months, and each one lasted an average of 9 days. This
means that they were sick for 1/3 of the time they spent at the day care centre, a
proportion consistent with our earlier findings (Uhari & Mottonen 1999).
We did not see any major changes in the colonic bacterial flora in our
children receiving cranberry juice when analysed in terms of the fatty-acid
composition of the stool (Kontiokari et al. 2005). This is a crude analysis of the
gram-negative bacterial flora of the gut and thus does not indicate that there
cannot be changes in the ability of the bacteria to cause UTI.
Cranberry juice at a dose of 5 ml/kg was well accepted by the children, and
their compliance was as good or better than with the long-term antimicrobial
regimen prescribed for UTI prophylaxis (Uhari et al. 1996b, Craig et al. 2009).
There was no single reason compromising its use. The children and their families
easily adopted the regimens of doses one to three times a day for several months,
so that in this sense cranberry juice is a feasible alternative for preventing UTIs.
82
83
8 Conclusions
1. A substantial proportion of uropathogenic E. coli strains are capable of
forming a biofilm in vitro, and the strains isolated from patients having
pyelonephritis form a biofilm better than those from cystitis cases. Given that
a biofilm protects bacteria from antibiotics, the ability of bacteria to persist
and grow in a biofilm seems to be one of the significant factors in the
pathogenesis of UTIs, and it should be taken into account when creating
strategies for prevention of UTIs.
2. In the absence of serious congenital anomalies, the aetiological fraction of
childhood UTIs as a cause of CKD after the first UTI in childhood is very
small. A child with normal kidneys has no significant risk of developing CKD
because of UTIs. Imaging procedures after the first UTI can be focused on
finding severe urinary tract abnormalities, for which purposes
ultrasonography would be sufficient.
3. Dietary elements are significant factors in susceptibility to UTIs. Taking
account of the relatively innocent nature of UTI recurrences in children who
do not have any marked urinary tract pathology, cranberry juice is a feasible
alternative to antimicrobials for preventing UTI in children. Cranberry juice
is well tolerated and accepted by children, and it does not cause harmful
changes in the normal flora.
84
85
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103
Original publications I Salo J, Sevander J, Tapiainen T, Ikäheimo I, Pokka T, Koskela M & Uhari M (2009)
Biofilm formation by Escherichia coli isolated from patients with urinary tract infections. Clinical Nephrology 71: 501–7
II Salo J, Ikäheimo R, Tapiainen T & Uhari M (2011) Childhood urinary tract infections as a cause of chronic kidney disease. Pediatrics 128: 840–7
III Salo J, Uhari M, Helminen M, Korppi M, Nieminen T, Pokka T & Kontiokari T (2012) Cranberry Juice for the Prevention of Recurrences of Urinary Tract Infections in Children: A Randomized Placebo-Controlled Trial. Clin Infect Dis 54: 340–6
IV Kontiokari T, Salo J, Eerola E & Uhari M (2005) Cranberry juice and bacterial colonization in children – a placebo-controlled randomized trial. Clin Nutr 24: 1065–72
Reprinted with permission from Dustri-Verlag (I), American Academy of
Pediatrics (II), Oxford Journals (III) and Elsevier (IV).
The original publications are not included in the electronic version of this
dissertation.
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LONG-TERM CONSEQUENCES AND PREVENTION OF URINARY TRACT INFECTIONSIN CHILDHOOD
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