Epidemiology of paediatric gastrointestinal colonisation by extended spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates in north-west Cambodia
1Nuffield Department of Clinical Medicineand the National Institute for Health Research Oxford Biomedical Research Centre (NIHR-OxBRC), University of Oxford, Oxford, United Kingdom
2Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom
3Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, UK
4Cambodia-Oxford Medical Research Unit, Angkor Hospitafor Children, Siem Reap, Cambodia
5Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK
6Angkor Hospital for Children, Siem Reap, Cambodia
7Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
#Corresponding author: Department of Microbiology/Infectious Diseases, John Radcliffe Hospital, Headley Way, Headington,OX3 9DU, United Kingdom. Tel: +44-(0)1865 220856; E-mail:nicole.stoesser@ndm.ox.ac.ukABSTRACT
Extended-spectrum cephalosporin resistance (ESC-R) in Escherichia coli and Klebsiella pneumoniae is a healthcare threat; high gastrointestinal carriage rates are reported from South-east Asia. Colonisation prevalence data in Cambodia are lacking. We determined gastrointestinal colonisation prevalence of ESC-resistant E. coli (ESC-R-EC) and K. pneumoniae (ESC-R-KP) in Cambodian children/adolescents and associated risk factors; characterised relevant resistance genes, their genetic contexts, and the genetic relatedness of ESC-R strains using whole genome sequencing (WGS). Faeces and questionnaire data were obtained from individuals <16 years in northwestern Cambodia, 2012. WGS of cultured ESC-R-EC/KP was performed (Illumina). Maximum likelihood phylogenies were used to characterise relatedness of isolates; ESC-R-associated resistance genes and their genetic contexts were identified from de novo assemblies using BLASTn and automated/manual annotation. 82/148 (55%) of children/adolescents were ESC-R-EC/KP colonised; 12/148 (8%) were co-colonised with both species. Independent risk factors for colonisation were hospitalisation (OR: 3.12, 95%, CI [1.52-6.38]) and intestinal parasites (OR: 3.11 [1.29-7.51]); school attendance conferred decreased risk (OR: 0.44 [0.21-0.92]. ESC-R strains were diverse; the commonest ESC-R mechanisms were blaCTX-M 1 and 9 sub-family variants. Structures flanking these genes were highly variable, and for blaCTX-M-15, -55 and -27, frequently involved IS26. Chromosomal blaCTX-M integration was common in E. coli. Gastrointestinal ESC-R-EC/KP colonisation is widespread in Cambodian children/adolescents; hospital admission and intestinal parasites are independent risk factors. The genetic contexts of blaCTX-M are highly mosaic, consistent with rapid horizontal exchange. Chromosomal integration of blaCTX-M may result in stable propagation in these community-associated pathogens.
MAIN TEXT
INTRODUCTION
Escherichia coli and Klebsiella pneumoniae are two bacterial pathogens of the Enterobacteriaceae family that can cause a wide spectrum of clinical disease, ranging from cystitis and intra-abdominal abscesses to sepsis. Both species also asymptomatically colonise the gastrointestinal tract, a reservoir that assists in the acquisition and spread of antimicrobial resistance (AMR)(1, 2). The increasing prevalence of AMR worldwide is reducing the efficacy of our limited armamentarium of empirical broad-spectrum antibiotics, such as extended-spectrum cephalosporins (ESCs), resulting in increased healthcare costs and mortality(3-5).
Recent reports from South-east Asia show substantial variation between country and cohort in gastrointestinal colonisation by Enterobacteriaceae possessing Ambler class A extended spectrum beta-lactamases (ESBLs) and/or class C AmpC enzymes, which can hydrolyse third and fourth generation cephalosporins. In the Lao People‘s Democratic Republic, for example, 23% of pre-school children carried these strains, in contrast to a much higher prevalence of 65.7% in a rural Thai adult population(6-8). Data describing the prevalence and mechanisms of antibiotic resistance in Cambodia are limited to only a few studies. Vlieghe and colleagues found 49.7% of Enterobacteriaceae from blood cultures in Phnom Penh from 2007-2010 were cefotaxime-resistant, mostly due to CTX-M-15 and CTX-M-14 enzymes(9). Studies from 2004/5 and 2007-2011 identified ESC resistance in 36-44% of urinary tract infection isolates(10, 11). The gastrointestinal colonisation prevalence of ESC-resistant (ESC-R) E. coli and K. pneumoniae in Cambodia has previously only been investigated in hospitalised neonates(12).
This study aimed to: (i) estimate the prevalence of gastrointestinal colonisation with ESC-resistant E. coli (ESC-R-EC) and K. pneumoniae (ESC-R-KP) in Cambodian children and adolescents, and the molecular mechanisms responsible; (ii) investigate risk factors for ESC-R colonisation; (iii) determine genetic relatedness of ESC-R strains.
RESULTS
Sampling, culture and basic demographics
In total, 196 faecal samples were obtained from a consecutive subset of children/adolescents enrolled in a helminth prevalence study. 48 samples were excluded from this study because of: (i) lack of specific consent for wider use of the faecal samples beyond the helminth survey (n=36); (ii) no epidemiological data records (n=1); (iii) no (n=3) or poor (n=5) growth on culture; or (iv) replicate samples for the same patient (n=3), leaving 148 samples/individuals for analysis.
Overall, 184 distinct colony types grew within the cefpodoxime inhibition zones; 141 were pink (presumed E. coli) and 43 were blue (presumed Klebsiella spp., Enterobacter spp. or Citrobacter spp.). All pink colonies but only 22/43 (54%) blue colonies were confirmed as phenotypically ESC-R using BSAC methods. All 163 confirmed ESC-R isolates were sequenced; two failed and were excluded from further analysis. Of the 161 sequences, in silico species identification confirmed 135 (84%) isolates were E. coli, 18 (11%) K. pneumoniae, and 8 (5%) Enterobacter spp. 38 E. coli isolates and one K. pneumoniae isolate were genetically sufficiently closely related to another isolate obtained from the same patient sample to be considered as the same strain (defined as ≤5 chromosomal SNVs); these were also excluded leaving 122 isolates for analysis. None of the 148 faecal samples yielded imipenem resistant colonies.
Participants were median 4.2 years old (interquartile range: 1.1-8.8) at sample collection; 70/148 (47%) were male. 70/147 (48%; 1 missing) were inpatients at sample collection. Although most were from Siem Reap province (99/148 [67%]), the hospital catchment is such that the remainder were recruited from 10 other provinces. 16/148 (11%) were clinically malnourished, and 23/148 (16%) had ≥1 underlying chronic medical condition including HIV (n=5), haematological disease (n=3), congenital cardiac disease (n=5), tuberculosis (n=4), and asthma (n=2)(Table 1).
Prevalence of and risk factors for colonisation with ESC-R EC and/or ESC-R-KP
A total of 114 confirmed ESC-R-EC (n=97) and ESC-R-KP (n=17) remained in the analysis and were carried by 82/148 participants, giving a combined ESC-R-EC/KP prevalence of 55% (95% CI: 47%-64%); 53% for ESC-R EC (79/148 patients; 95% CI: 45%-62%) and 10% for ESC-R KP (15/148 patients; 95% CI: 6%-16%). Co-colonisation with both ESC-R-EC and ESC-R-KP was observed in 12/82 (15%). Independent risk factors for ESC-R-EC/KP colonisation included being a current inpatient (OR=3.64; 95% CI [1.71-7.74), p=0.001) and the presence of faecal parasites (OR=3.96 [1.55-10.13], p=0.004). ESC-R-EC/KP colonisation was lower in males (OR=0.39 [0.18-0.84], p=0.015) and in those attending school (OR=0.39 [0.18-0.83], p=0.015)(Table 1).
Sequence type, Ambler class and genetic mechanisms of ESC-R
The 97 ESC-R-EC isolates came from 33 known and 6 novel STs (Fig.1, for details see Table S1). 22% (17/79) of patients were colonised by at least two different ESC-R-EC STs, although this may underestimate diversity as only a small number of colonies (≤3) were sampled per patient(25). The 17 ESC-R-KP strains came from 11 known and 3 novel STs (n=4 isolates) (Fig.2, Table S2). Two patients were colonised by two different ESC-R K. pneumoniae STs (2/15, 13%).
In total, 77% (88/114) and 23% (26/114) of isolates displayed Ambler class A or C phenotypes, respectively. Neither species were associated with Ambler class A (76% [74/97] versus 82% [14/17]) or class C (23% [23/97] versus 18% [3/17]; Fishers exact test; p=0.759). In all class A isolates the phenotype could be explained by the presence of one (84/88, 95%) or two (4/88, 5%) blaCTX-M genes; blaSHV (12/88, 12%) and blaVEB (1/88, 1%) occurred less commonly. Class C gene families were only identified in 39% (10/26) of phenotypically class C isolates: specifically blaCMY-2 (8/26, 31%) or blaDHA (2/26, 8%). In the remaining 16 isolates, the genetic basis for the class C phenotype was unclear; of note, however, ampC promoter mutations were not assessed. 111 blaCTX-M genes were found in 94% (107/114) of ESC-R-EC/KP, with two separate alleles identified in 4% of isolates (4/114). The most frequently identified allele was blaCTX-M-15 (53/111, 48%), followed by: blaCTX-M-55 (24/111, 22%), blaCTX-M-14 (17/111,15%), blaCTX-M-27 (14/111, 13%) and blaCTX-M-24 (3/111, 3%). Two different blaCTX-M alleles were found in 21% (18/82) of individuals carrying ESC-R-EC/KP. blaSHV genes were identified in 15/17 K. pneumoniae, including blaSHV-1/ SHV-1-like (3/15, 20%), blaSHV-11/ SHV-11-like (4/15, 27%), blaSHV-27-like (1/15, 7%), blaSHV-28 (1/15, 7%), blaSHV-33 (3/15, 20.0%) and blaSHV-83 (1/15, 7%), blaSHV-99-like (1/15, 7%) and blaSHV-142 (1/15, 7%). All eight blaCMY-2/CMY-2-like genes were found in E. coli. The study population carriage prevalence of common genetic mechanisms encoded by ESC-R EC/KP was therefore: 53% blaCTX-M (78/148), 9% blaSHV (14/148), 1% blaVEB (1/148), 5% blaCMY-2 (8/148), 1% blaDHA (2/148). Two individuals (1%) carried isolates with blaOXA-48 (one K. pneumoniae ST48 and one E. coli ST648); no other carbapenem resistance mechanisms were identified.
Genetic context of ESC-R genes
For the 41 E. coli harbouring blaCTX-M-15, it was chromosomally located in five cases (12%), and likely in plasmid contexts in two; in the remaining cases it was not possible to determine wider chromosomal/plasmid location (Table 2). One isolate (38P1) harboured short contigs containing truncated blaCTX-M-15, leaving 40 cases in which to evaluate the immediate flanking contexts surrounding the blaCTX-M gene. All contained ISEcp1 upstream of blaCTX-M- 15, but with considerable evidence of additional mobilisation events/mosaicism (Table 2). In particular, ISEcp1 was truncated by IS26 at 24, 497, 524, 1067, 1173, 1421, or 1489bp in 13 isolates, consistent with at least seven IS26-associated insertion events within ISEcp1(Fig.3). Another 13 ISEcp1 elements were truncated by contig breaks, without any specific associated genetic signatures, although contig breaks are frequently due to repeat structures and may therefore have represented additional disruption events. One isolate had an intact ISEcp1 element, without any wider flanking upstream context. The 13 cases with an intact ISEcp1 were consistently flanked by variable lengths of Tn2, which was truncated by an IS26 right IRR in 2/7 evaluable cases (and by an unknown sequence in the other 5/7). Two isolates had a complete Tn2 structure interrupted by ISEcp1-blaCTX-M-15 (TCTCA-TCTCA and TTTTA-TAAAA target site sequences [TSSs] respectively)(Fig.3). Overall, genetic contexts of blaCTX-M-15 were consistent with integration and mobilisation of ISEcp1-blaCTX-M-15 within a Tn2 element, as previously described(26), with subsequent rearrangement events facilitated by IS26 and perhaps other ISs(27)(Table S3).
For the 24 E. coli harbouring blaCTX-M-55, it was chromosomally located in 4 (17%), plasmid in 3 (13%) and unknown in 16 (67%). One contig contained a truncated blaCTX-M-55, leaving 23 evaluable contexts. Similar to blaCTX-M-15, it was invariably associated with ISEcp1 upstream of blaCTX-M-55 (Fig.4), which was often incomplete, representing at least 3 different IS26-associated ISEcp1 disruption events (Table 2). Intact ISEcp1 were flanked by variable lengths of Tn2 sequence, apart from 120P1 where the contig was truncated immediately at the 5‘ end of ISEcp1. One isolate (2P1) had the same blaCTX-M/Tn2 unit as for blaCTX-M-15 (but with TACTC-TAAAA), consistent with the evolution of blaCTX-M-55 from blaCTX-M-15 (1 SNV difference) within this unit (Figs.3, 4).
For the 15 E. coli harbouring blaCTX-M-14, it was chromosomally located in 2 (13%) cases, plasmid-associated in 5 (33%), and unknown in 8 (53%). Again, it was invariably associated with ISEcp1, but more often complete and with different mechanisms of disruption (2 ISVsa5-like sequence, one IS1S R IRR). All cases had an IS903 element at the 3‘ end of blaCTX-M-14; this had been disrupted in 6 cases, with additional contig breaks in 5 cases (Fig.5). Two of three E. coli blaCTX-M-24 contexts were chromosomal, with flanking contexts similar to blaCTX-M-14 (Fig.S1). In the 12 blaCTX-M-27 cases, the ISEcp1 element had been disrupted by an IS26 L IRR in all contexts, at 149, 192, 208 and 388bp, but the wider genetic context of this structure was indeterminable in all cases (Fig.S2).
Overall, blaCTX-M was chromosomal in 13/92 cases (14%; 13/25 [52%] cases where plasmid versus chromosomal location could be assessed), suggesting that CTX-M genes may be incorporated chromosomally and indiscriminately in significant numbers of colonising E. coli, with possible implications for their stable propagation within the wider E. coli population.
For K. pneumoniae, 12 isolates harboured blaCTX-M-15, in a plasmid-associated context in 9/12 cases, and an unknown context in 3/12 cases. Three isolates harboured a complete blaCTX-M-15 /Tn2 complex with GTTAA-GTTAA TSS, most consistent with a direct transposition of this element into a plasmid context. In the other isolates, the ISEcp1-blaCTX-M-15-ORF477 was flanked by variable stretches of Tn2-associated sequence identical to that found in the E. coli isolates, and similarly truncated either as a result of contig breaks, or by IS26 inverted repeats, consistent with between species and within species mobilisation (Fig.S3).
Four K. pneumoniae isolates harboured blaCTX-M-9 group genes; two of these (blaCTX-M-14) shared the same ISEcp1 (Fig.5) and ∼18kb upstream flanking plasmid sequence; and two (blaCTX-M-27) an ISEcp1 element truncated at position 1499 by an IS26 L IRR (Fig.S2).
DISCUSSION
We observed significant gastrointestinal carriage prevalence of both ESC-R-EC and ESC-R-KP in Cambodian children sampled in 2012; approximately one in twelve children was co-colonised with ESC-R strains of both species. A wide diversity of ESC-R strain types was observed, including several genotypes categorised as “high risk” clones, such as E. coli STs 38, 405, 131, 354 and 648(13). The predominant ESC-R genotypic mechanism was blaCTX-M, with the major allelic variants being those widely described elsewhere in Asia (Group 1: blaCTX-M-15, -55, Group 9: blaCTX-M-14, -24, -27). Approximately one-third of the Cambodian population is <18 years old, so this group may be acting as a significant reservoir for the spread of anti-microbial resistant organisms. We did not observe particularly high rates of colonisation with carbapenem-resistant isolates (2 (1%) individuals), but one of these was an out-patient, with an OXA-48 E. coli isolate, and without any known chronic health problems, suggesting that there may be some carriage of carbapenem-resistant isolates in the community. Further assessment of the extent of carriage of carbapenem-resistant EC/KP in this context is warranted.
Independent risk factors for colonisation included inpatient status, consistent with transmission within hospital, and/or selection of these organisms from low-level carriage by the use of antibiotics on admission given the high burden of infectious diseases in this region. Infection control (IC) in resource-limited settings remains challenging, and despite improvements within the study hospital(14), recent longitudinal surveillance within the neonatal care unit identified high rates of import of ESC-R-KP (62% colonised on admission) as well as nosocomial acquisition (23%)(12). In-patient acquisition of ESC-R-EC/KP has also been identified as a major problem in other low/middle-income settings(15). The specific effect of faecal parasites on gut microbiota is not well-studied, but they are thought to significantly perturb microbial diversity(16). Helminth infestation may also result in inappropriate antimicrobial use, including antibiotics, perhaps leading to secondary colonization with drug-resistant commensals. The decreased risk associated with school attendance has been observed in a previous study in Spain(17), and may represent a proxy marker for increased socio-economic status, and parental levels of education, which were not evaluated here, but may translate into better awareness of appropriate antibiotic use(18, 19). The decreased risk associated with male gender is unexplained; but independent associations for ESBL-EC/KP colonization have been described for both genders in previous studies(15, 20, 21).
Of particular importance was the high prevalence of chromosomal integration of blaCTX-M in E. coli in this study (>14%), perhaps contributing to the stable propagation of this resistance gene family within certain strains. Chromosomal integration of blaCTX-M in K. pneumoniae was not observed in our study, although it has been seen in Spain(22). In addition, despite the limitations of short-read assemblies, the genetic contexts of blaCTX-M suggested high levels of genetic plasticity in flanking structures, and significant associations with IS26 for blaCTX-M-15, blaCTX-M-55, and blaCTX-M-27. IS26 has been previously hypothesised to facilitate the mobility of blaCTX-M and genetic rearrangement of resistance gene plasmids, and is likely contributing to the dissemination of these resistance genes within the human gastrointestinal reservoir(23-25).
This study has several limitations. Our survey dates from 2012, and the epidemiology of ESC-R EC/KP carriage may have changed in the intervening timeframe; nevertheless, our data represent the largest molecular epidemiological study of gastrointestinal ESC-R-EC/KP colonisation in Cambodia and a useful benchmark for future studies. We only included up to three bacterial colonies per faecal sample, likely resulting in significant under-estimation of the diversity present at the population level(26). Short-read sequencing resulted in limited information regarding the wider genetic context of important resistance genes conferring ESC-R; nevertheless, we were still able to ascertain that the genetic contexts of these resistance genes are extremely diverse. Our outpatient study population may not be truly representative of healthy children in the community, given that these individuals had presented to the outpatient department for some form of medical review. Lack of more detailed information on some potential risk factors meant we were unable to fully assess the specific mechanisms promoting ESC-R EC/KP colonisation. Further work characterising the role of healthcare admissions, socio-economic factors and intestinal parasites on the acquisition and long-term carriage dynamics of these strains would be valuable. In addition, our sample size was too small and sparse to investigate geographical clustering of strain types, and to investigate specific risk factors for colonisation with common strain types or resistance gene alleles.
Despite these limitations, our study adds to the growing body of literature demonstrating widespread gastrointestinal colonisation with ESC-R-EC and ESC-R KP in Southeast Asia(8), and showing that exposure to this reservoir may in turn act as a source for the wider, global transfer of these strains(27). The genetic contexts of important resistance genes are highly mosaic, consistent with rapid exchange of resistance genes within and between bacterial hosts. Significant levels of chromosomal integration of the most important ESC-R gene family, blaCTX-M, were also observed, and may result in these genes being stably maintained and propagated in one of the most common community-associated pathogens, namely E. coli.
MATERIALS AND METHODS
Patients and setting
Faecal samples were obtained from a consecutive subset of children/adolescents (<16 years) who had been enrolled prospectively in a helminth prevalence study at Angkor Hospital for Children in Siem Reap, Cambodia, from 3rd April 2012 to 29th June 2012, as described in(28).
Microbiological methods
Samples were frozen at -80°C as aliquots homogenised in 0.9% sterile saline with 10% glycerol within an hour of receipt in the laboratory. For this study, faecal samples were thawed, and aliquots diluted 1:10 in saline and incubated for 16 hours at 37°C on Orientation CHROMagar (BD, Oxford, United Kingdom) with 10 µg cefpodoxime and 10 µg imipenem discs (Oxoid, Basingstoke, United Kingdom). For each faecal sample, up to three pink and/or dark blue colonies with different colonial morphotypes that grew within the cefpodoxime zone of inhibition (presumed ESC-R-EC and ESC-R-KP respectively) were selected for further analysis. Each selected colony was tested using the British Society of Antimicrobial Chemotherapy (BSAC) combination disc method to identify whether cefpodoxime (ESC) resistance was mediated via ESBLs (Class A: cefpodoxime-resistant, and cefpodoxime+clavulanic acid-sensitive) or via non-ESBL mechanisms (e.g. Class C AmpC beta-lactamases: cefpodoxime-resistant, and cefpodoxime+clavulanic acid-resistant)(29). All identified ESC-R colonies were stored frozen at -80°C in nutrient broth with 10% glycerol.
Whole genome sequencing and sequence data processing
DNA was extracted from sub-cultured ESC-R isolates using a commercial kit (Fujifilm Quickgene, Japan) with an additional mechanical lysis step (Fastprep MP Biomedicals, USA). All isolates were sequenced using the Illumina HiSeq 2500, generating 150bp paired-end reads. Sequence data have been deposited in GenBank (project accession: PRJNA391054).
To identify single nucleotide variants (SNVs) reads were mapped to species-appropriate reference genomes (E. coli CFT073 [GenBank: AE014075.1] and K. pneumoniae MGH78578 [GenBank: CP000647.1]), and variants called as described previously(30). Alignments of variable sites were padded to the length of the reference genome using bases with the same %GC content as that observed within each dataset. Bootstrapped, maximum-likelihood phylogenies were reconstructed for each species using RaxML version 7.7.6(31), using a generalised time-reversible model and four categories of rate heterogeneity (./RAxML-7.7.6/raxmlHPC-PTHREADS-SSE3 -f a -s <input_alignment.phy> -m GTRGAMMA -p 12345 -c 4 -x 12345 -# 100 -n <output_raxml_rapid_bootstrap>). Phylogenies have been deposited as projects in MicroReact to enable an interactive assessment of geographic distribution of genotypes (E. coli: https://microreact.org/project/By8bf5ajg; K. pneumoniae: https://microreact.org/project/Hy_yQcaog(32).
Contigs were assembled using Velvet/VelvetOptimiser (hash value range: 75-149)(33, 34). In silico MLST was determined by BLASTn(35) matches (100% match) to the Achtman/Pasteur MLST schemes for E. coli and K. pneumoniae(36, 37), and supported correct species identification. The presence/absence of resistance genes was determined using BLASTn and an in-house curated resistance gene database of over 60 gene families(38). Genes were considered present if a blast match of ≥80% of the query sequence was identified at ≥80% sequence identity using the de novo assemblies as blast databases. Ambler class genotype was class A if blaCTX-M, blaSHV and/or blaVEB were present, and/or class C if blaCMY-2, blaDHA and blaACT-like genes were present. Where patient faecal samples yielded ≥2 strains, all resistance genes were treated as a single entity within the individual‘s profile.
The genetic context of blaCTX-M was examined by extracting the contigs containing these genes, and annotating these using PROKKA(39), combined with BLASTn and manual annotation with reference to mobile genetic elements in the ISFinder database(40). Gene locations were characterised as “chromosomal” if other annotations on the contig were only found in chromosomal contexts in the top 20 BLASTn hits when the contig was compared with bacterial sequences available in GenBank (using default parameters); “plasmid” if the other annotations matched only plasmid sequences; or unknown if these conditions were not met e.g. the assembled contigs were too short to verify this.
Epidemiological analyses
Information regarding putative risk factors for ESC-R EC/KP colonisation (collected on a standardised form) included details on: gender, age, hospitalisation status, residence in Siem Reap province versus elsewhere, water source (river, rain, well, bottled, piped, boiled), domestic animals (cats, dogs, birds), livestock (chickens, ducks, pigs, cows or water buffalo), toilet availability, malnutrition, co-morbidities, presence/absence of diarrhoea, presence/absence of parasites (assessed within (28)), soap usage for hand-washing and school attendance. No details regarding antibiotic consumption were ascertained within the study, but previous work locally has shown that individuals are often ill-informed about the nature of any medications used and that 32% of outpatient attendees have evidence of urinary antimicrobial activity(41).
Statistical analyses
Independent risk factors for carriage were identified from a multivariable, stepwise, logistic regression model based on complete cases and initially including all factors (backwards elimination using exit p<0.1 to reduce over-fitting). A final multivariable logistic model was then fitted including all cases for which complete information was available for the retained risk factors. Statistical analyses were performed using STATA version 14 (StataCorp, College Station, USA).
Ethical Approval
The study was approved by the Institutional Review Board (IRB), Angkor Hospital for Children, and the Oxford Tropical Research Ethics Committee (OXTREC 12–12). Caregivers of all included participants gave informed consent for their child to participate in the helminth survey, and for the samples to be used more widely in additional studies approved by the IRB.
Acknowledgements
The authors wish to thank the staff and patients at Angkor Hospital for Children, Siem Reap, Cambodia, and members of the Modernising Medical Microbiology Informatics Group.
This work was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Center (BRC). JvA is currently funded through a National Institute for Health Research (NIHR) Academic Clinical Fellowship. NS is currently funded through a PHE/NIHR/University of Oxford Clinical Lectureship; the sequencing work was also partly funded through a previous Wellcome Trust Doctoral Research Fellowship (#099423/Z/12/Z). TEAP and DWC are NIHR Senior Investigators.
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.
The authors have no conflicts of interest to declare.