Transactions of the Royal Society of Tropical Medicine and Hygiene

Transactions of the Royal Society of Tropical Medicine and Hygiene 106 (2012) 718 724 Contents lists available at SciVerse ScienceDirect Transactions of the Royal Society of Tropical Medicine and Hygiene j our na l ho me p age: htt p: //www.elsevier.com/locate/trstmh in Cambodian children is dominated by multidrug-resistant H58 Salmonella enterica serovar Typhi with intermediate susceptibility to ciprofloxacin Kate Emary a,b, Catrin E. Moore a,b,c, Ngoun Chanpheaktra c, Khun Peng An c, Kheng Chheng c, Soeng Sona c, Pham Thanh Duy d, Tran Vu Thieu Nga d, Vanaporn Wuthiekanun a, Premjit Amornchai a, Varun Kumar c, Lalith Wijedoru a,b, Nicole E. Stoesser a,b, Michael J. Carter a,b,e, Stephen Baker b,d, Nicholas P.J. Day a,b, Christopher M. Parry a,b,c, a Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand b Centre for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford OX3 9DU, UK c Angkor Hospital for Children, Siem Reap, Kingdom of Cambodia d Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Vo Van Kiet, District 5, Ho Chi Minh City, Vietnam e Institute of Child Health, University College London, London WCIN 3EH, UK a r t i c l e i n f o Article history: Received 17 May 2012 Accepted 7 August 2012 Available online 1 November 2012 Keywords: Salmonella Typhoid Antimicrobial resistance Treatment Complications Cambodia a b s t r a c t Infections with Salmonella enterica serovar Typhi isolates that are multidrug resistant (MDR: resistant to chloramphenicol, ampicillin, trimethoprim sulphamethoxazole) with intermediate ciprofloxacin susceptibility are widespread in Asia but there is little information from Cambodia. We studied invasive salmonellosis in children at a paediatric hospital in Siem Reap, Cambodia. Between 2007 and 2011 Salmonella was isolated from a blood culture in 162 children. There were 151 children with enteric fever, including 148 serovar Typhi and three serovar Paratyphi A infections, and 11 children with a non-typhoidal Salmonella infection. Of the 148 serovar Typhi isolates 126 (85%) were MDR and 133 (90%) had intermediate ciprofloxacin susceptibility. Inpatient antimicrobial treatment was ceftriaxone alone or initial ceftriaxone followed by a step-down to oral ciprofloxacin or azithromycin. Complications developed in 37/128 (29%) children admitted with enteric fever and two (1.6%) died. There was one confirmed relapse. In a sample of 102 serovar Typhi strains genotyped by investigation of a subset of single nucleotide polymorphisms, 98 (96%) were the H58 haplotype, the majority of which had the common serine to phenylalanine substitution at codon 83 in the DNA gyrase. We conclude that antimicrobial-resistant enteric fever is common in Cambodian children and therapeutic options are limited. 2012 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. 1. Introduction Corresponding author. Present address: Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. Tel.: +66 02 203 6333; fax: +66 02 354 9169. E-mail address: chrisp@tropmedres.ac (C.M. Parry). Invasive bacterial infections are a significant cause of morbidity and mortality among children in southeast Asia. 1,2 Members of the genus Salmonella, including the enteric fever serovars Typhi and Paratyphi A, and various non-typhoidal serovars are commonly isolated from the 0035-9203/$ see front matter 2012 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.trstmh.2012.08.007

K. Emary et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 106 (2012) 718 724 719 blood of febrile children in resource-limited settings. 3 5 Isolates of serovar Typhi and Paratyphi A resistant to multiple antimicrobial agents have caused epidemics and are endemic in many areas of southeast and south Asia. 6 These include multidrug-resistant (MDR) isolates resistant to the previous first-line antimicrobials (chloramphenicol, ampicillin, co-trimoxazole) and those with intermediate susceptibility to ciprofloxacin (previously described as decreased ciprofloxacin susceptibility). 7,8 Antimicrobial resistance has restricted the treatment choice for enteric fever and other invasive salmonellosis. 6 In 2010 the under-five year mortality rate in the Kingdom of Cambodia was 54/1000 live births and the prevalence of malnutrition (below 2 SD of weight for age) was 28%. 9 There is little published information concerning invasive bacterial infections in Cambodian children, including typhoid fever, partly as a consequence of the lack of functional microbiology laboratories. 2 In a prospective community study conducted between 2006 and 2009 in children and adults with fever in two districts within 50 km of the capital, Phnom Penh, of almost 5000 blood cultures, S. enterica Typhi was isolated in 41 (0.9%). 10 Of these 41 serovar Typhi isolates, 23 (56%) were MDR and 36 (88%) had intermediate susceptibility to ciprofloxacin. At Angkor Hospital for Children (AHC), in Siem Reap province in northwest Cambodia, a microbiology laboratory with the capacity to perform blood cultures was established in 2006. Since April 2010 the laboratory has also received blood cultures from a satellite clinic and ward in Sotr Nikom District, 30 km away. A small study at AHC conducted in 2009 suggested that antimicrobialresistant enteric fever could be an important problem at this location. 11 Here we report a retrospective analysis of the demographic, epidemiological and clinical characteristics of children at these hospitals with Salmonella-positive blood cultures, between 2007 and 2011. 2. Materials and methods 2.1. Study population The study was conducted at AHC and the AHC Satellite Clinic (SC) in Sotr Nikom District, Siem Reap Province, Cambodia. The two facilities are charity funded and provide free outpatient, inpatient, emergency, surgical, medical, ophthalmological and dental care. There are 50 inpatient beds at AHC and 20 at SC and the two outpatient departments see more than 400 children each day from an unrestricted catchment area, with the majority of patients from Siem Reap or neighbouring provinces. This was a retrospective cross-sectional study of children from whom Salmonella was isolated from a blood culture between 1 January 2007 and 31 December 2011 inclusive. Children were identified by inspection of the laboratory register. A retrospective review of case records of identified children was performed to collect data on age, gender, clinical features, antimicrobial therapy (type, route and duration), infection-associated complications, duration of hospital stay and outcome at hospital discharge, all of which were documented on a standard case report form. Epi Info V.3.5.3 (CDC, Atlanta, GA, USA) was used to calculate the weight for age z scores using CDC Clinical Growth Charts for the United States (2000) (http://www.cdc.gov/growthcharts/). 2.2. Microbiological methods Blood cultures in children with fever or suspected sepsis were performed at the discretion of the clinicians. Blood for culture (generally between 1 and 2 ml) was collected by a member of the laboratory staff and inoculated into a bottle containing 20 ml Tryptic Soy Broth with added sodium polyethanolsuphonate (all media from Oxoid, Basingstoke, Hampshire, UK). The bottles were incubated at 35 37 C and inspected daily. If the broth was cloudy a Gram stain was performed and the broth inoculated onto sheep blood agar, chocolate agar and MacConkey agar. Otherwise a blind subculture was performed onto sheep blood and chocolate agar after 24 h, 48 h and 7 days of incubation. Gram-negative bacilli were identified by biochemical testing (triple sugar iron agar, motility, lysine decarboxylase, indole production, citrate and urea utilization) or API 20E (biomérieux, Marcy l Etoile, France). Putative S. enterica isolates were confirmed by agglutination with specific antisera (Bio-Rad, Hemel Hempstead, Hertfordshire, UK). Antimicrobial susceptibilities were performed at the time of isolation by a modified Bauer-Kirby disc diffusion method, inhibition zone sizes were recorded and interpretations of the zone sizes were based on the latest CLSI guidelines. 12 The antimicrobials tested (Oxoid) were chloramphenicol (30 g), ampicillin (10 g), trimethoprim sulphamethoxazole (1.25/23.75 g), ceftriaxone (30 g), ciprofloxacin (5 g), azithromycin (15 g) and nalidixic acid (30 g). Isolates were stored in Tryptic Soy Broth with 20% glycerol at 80 C. A representative selection of 102 stored S. enterica Typhi isolates were later subcultured and the minimum inhibitory concentration (MIC) determined by E-test strips according to the manufacturer s guidelines (AB Biodisk, Solna, Sweden). The evaluated antimicrobials were ciprofloxacin, gatifloxacin, ceftriaxone and azithromycin. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as control strains for these assays. An isolate was defined as MDR if it was resistant to all of the following: chloramphenicol ( 32 g/ml), ampicillin ( 32 g/ml) and trimethoprim/sulfamethoxazole ( 8/152 g/ml). Intermediate susceptibility to ciprofloxacin (formerly known as decreased ciprofloxacin susceptibility) was defined by an MIC of 0.12 0.5 g/ml and resistance by an MIC of 1 g/ml. 12 The equivalent values for gatifloxacin were 4 g/ml and 8 g/ml and for ceftriaxone 2 g/ml and 4 g/ml. There are no recommended CLSI breakpoints for azithromycin against Salmonella. 2.3. Molecular typing We sought to distinguish the H58 serovar Typhi strains, as these are the most common and ubiquitous across Asia, from non-h58 strains by inferring genotype though the detection of the H58-specific single nucleotide

720 K. Emary et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 106 (2012) 718 724 polymorphism (SNP) using a modified pyrosequencing technique. Salmonella Typhi belong to haplotype H58 if the SNP at nucleotide 252 on the gene glpa (corresponds to STY2513 from GenBank accession no. AL513382, Salmonella Typhi CT18) is T, otherwise they belong to non-h58. 13 The common SNPs inducing intermediate susceptibility to ciprofloxacin, located at position 83 and 87 in the gyra gene and position 80 in the parc gene, were also determined by modified pyrosequencing. 7 Genomic DNA was prepared from the bacterial isolates using the Wizard genomic DNA purification kit (Promega, Madison, WI, USA). The prepared DNA was PCR amplified using the following primer pairs targeting the regions containing the H58 SNP: forward primer 5 biotin GTAACGTCAGCCGCGGTATT; reverse primer 5 GCCATCAGGCGATAAGTCATTA 3. SNPs in the gyra gene (codons 83 and 87) and a SNP in the parc (codon 80) gene were amplified and sequenced using the following primers: gyra codon 83: F 5 TGGGCAATGACTGGAACAAAG 3, R 5 biotin TACCGTCATAGTTATCCACG 3, Seq 5 CGGTAAAT- ACCATCCCCA 3 ; gyra codon 87: F 5 TGGGCAATGACTG- GAACAAAG 3, R 5 biotin TACCGTCATAGTTATCCACG 3, Seq 5 CGATTCCGCAGTGTA 3 ; and parc codon80: F 5 biotin ACCGTTGGCGACGTACTGG 3, R 5 AATTCCCCTGGCCATC- GAC 3, Seq 5 CATGGCTTCATAGCAG 3. All PCR amplifications were performed in 60 l reactions containing 1 NH 4 buffer, 1.5 mm of MgCl 2, 200 M of dntp, 10 pm of each primer, 1.25 units of Hotstart DNA polymerase (Qiagen, Valencia, CA, USA) and 5 l of template DNA. Reactions were cycled once at 95 C for 15 min, followed by 30 cycles of 94 C for 1 min, 55 C for 1 min and 72 C for 1 min, with a final elongation of 72 C for 5 min. All PCR amplifications were observed on 1% agarose gels prior to pyrosequencing to ensure correct amplification. We used a pyrosequencer (Pyromark Q96 ID DNA pyrosequencer, Biotage, Uppsala, Sweden) to detect the SNP in each of the loci sequenced. PCR amplicons were combined with 56 l of binding buffer and 4 l of streptavidin sepharose beads. The resulting mixture was vigorously agitated for 5 min before being denatured in denaturation buffer and washed using the Vacuum Prep Tool (Biotage). The single stranded DNA fragments were transferred into a 96-well plate containing 3.5 pmol of sequencing primer in 40 l of annealing buffer and the DNA sequencing reaction was performed using the Pyro Gold Kit (Qiagen). 2.4. Analysis Analysis of demographic and clinical data was descriptive with continuous variables described by a median and IQR and compared by the Mann Whitney U test. Proportions were compared using the 2 test or Fisher s exact test. Analysis including multivariate analysis of factors associated with complicated disease was performed using Epi Info (CDC) and SPSS V.18.0 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Patients During the 5-year study, Salmonella was isolated from the blood of febrile children on 162 occasions. One hundred and fifty-one children had enteric fever (148 with serovar Typhi and 3 with serovar Paratyphi A), of whom 128 (85%) were hospitalised and 24 (15%) were managed as outpatients. Eleven children had a non-typhoidal Salmonella (NTS) serovar isolated from the blood culture, 10 (91%) of these were hospitalised and one (9%) was managed as an outpatient. Table 1 Clinical features of Cambodian children admitted to hospital with culture-confirmed enteric fever or invasive non-typhoidal salmonellosis (NTS) between 2007 and 2011 Cases with enteric fever (n=128) Cases with NTS (n=10) Median (IQR) age (years) 7.0 (4.5 10.4) 0.9 (0.3 2.9) Male gender 63 (49) 7 (70) Weight for age z score < 3 49/126 (39) 4/8 (50) Median (IQR) prior duration of illness (days) 6 (5 10) 5 (3 7) Chills 42 (33) 0 Headache 54 (42) 0 Cough 37 (29) 4 (40) Vomiting 73 (57) 6 (60) Diarrhoea 46 (36) 7 (70) Constipation 17 (13) 0 Abdominal pain 103 (80) 0 Underlying disease 7 (5) 5 (50) Hepatomegaly 70 (55) 3 (30) Splenomegaly 11 (9) 0 Abdominal distension 31 (24) 1 (10) Abnormal mental state 27 (21) 3 (30) Peak temperature 40.0 C 68/126 (54) 0/8 Median (IQR) haemoglobin (g/dl) 9.3 (7.5 10.8) 9.8 (6.8 11.1) Anaemia (<7 g/dl) 73/125 (58) 6/9 (67) Median (IQR) white cell count 7.1 (5.3 9.6) 14.5 (10.0 28.3) Median (IQR) neutrophil count 4.3 (2.8 6.1) 7.2 (4.0 15.8) Median (IQR) lymphocyte count 2.0 (1.2 2.9) 6.1 (3.6 6.9) Median (IQR) platelet count 213 (142 311) 412 (125 677) Data are number (%), unless otherwise indicated.

K. Emary et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 106 (2012) 718 724 721 25 Non-typhoidal salmonellosis 20 No. of cases 15 10 5 0 <1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Age (years) Figure 1. Age distribution of children admitted with blood culture positive enteric fever or non-typhoidal salmonellosis between 2007 and 2011. 3.2. Clinical features The clinical features of the hospitalised children with invasive Salmonella are shown in Table 1 and the age distribution of the hospitalised children is shown in Figure 1. The median (IQR, range) age of the children with enteric fever was 7.6 years (4.9 11.2 years, 8 months 16 years), 38/151 (25.2%) were under the age of 5 years and 71/151 (47%) were male. The median (IQR, range) age of the children with NTS was 1 year (5 months 6.9 years, 1 month 12 years), 8/11 (73%) were under the age of 5 years and 8/11 (73%) were male. Five (45%) of the 11 NTS-infected children were known to be HIV seropositive (one under 1 year) and a further five (45%) were less than 1 year in age with bacteraemia secondary to diarrhoea. For the children with a weight recorded, 60/158 (38%) were malnourished, with a weight-for-age z score of 3, and 87/154 (56%) children with haemoglobin level available had a level below 10 g/dl. 3.3. Antimicrobial susceptibility patterns Eighty-five percent (126/148) of serovar Typhi isolates were MDR, 90% (133/148) had intermediate susceptibility to ciprofloxacin and 80% (119/148) were both MDR with intermediate susceptibility to ciprofloxacin. There was no significant variation in the proportion of strains that were MDR or with intermediate susceptibility to ciprofloxacin over the 5-year study period. None of the isolates were resistant to ciprofloxacin and all were susceptible to ceftriaxone. None of the three serovar Paratyphi A isolates were MDR but all had intermediate susceptibility to ciprofloxacin and 2/11 (18.2%) of the NTS isolates were MDR with 4/11 (36.4%) with intermediate susceptibility to ciprofloxacin. MICs were determined for a representative sample of 102 serovar Typhi isolates (Table 2) and the genotype was used to infer haplotype and mutations in the gyra and parc loci. Ninety-six percent (98/102) of the genotyped serovar Typhi isolates were haplotype H58. Furthermore, the majority of H58 isolates were accompanied by a single mutation or multiple mutations in the gyra gene. The majority, 93, exhibited the most described mutation, encoding an amino acid substitution from serine to phenylalanine at codon position 83 (S83F) in the DNA gyrase protein. Three isolates containing the S83F substitution were also accompanied by a mutation inducing a change from aspartic acid to glycine at position 87 (D87G), one isolate had a single substitution of aspartic acid to tyrosine at position 87 (D87Y) and three exhibited no mutations in gyra. The remaining four serovar Typhi isolates did not belong to the H58 haplotype, and had no mutations in the gyra gene and were susceptible to ciprofloxacin. The three serovar Paratyphi A isolates all had an aspartic acid to asparagine gyrase A substitution at position 87 (D87N). 3.4. Clinical course and outcome Various antimicrobial regimens were used for the children admitted to hospital with enteric fever (Table 3), with most children (126/128; 98%) initially treated with ceftriaxone. Ceftriaxone monotherapy was given to Table 2 Minimum inhibitory concentrations of Salmonella enterica Typhi isolates from blood in Cambodian children against four antimicrobials by the E-test strip method (n=102) Antimicrobial MIC 50 ( g/ml) MIC 90 ( g/ml) Minimum ( g/ml) Maximum ( g/ml) Ciprofloxacin 0.380 0.500 0.006 0.500 Gatifloxacin 0.190 0.250 0.008 0.380 Ceftriaxone 0.090 0.125 0.006 0.190 Azithromycin 12 16 6 16 MIC 50 and MIC 90: minimum inhibitory concentration of the antimicrobial required to inhibit 50% and 90% of the tested isolates, respectively.

722 K. Emary et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 106 (2012) 718 724 Table 3 Response to treatment according to antimicrobial regimen given in 128 Cambodian children admitted to hospital with enteric fever Antimicrobial regimen No. of children Median (IQR) duration of antimicrobial given (days) Median (IQR) fever clearance time (days) No. (%) with fever clearance >7 days No. (%) with complication Ceftriaxone 58 10 (7 14) 7.7 (4.0 10.8) 23 (47) 22 (38) Ceftriaxone then ciprofloxacin 25 14 (12 17) 6.4 (4.3 11.2) 9 (36) 2 (8) Ceftriaxone then azithromycin 42 13 (11 16) 4.4 (3.4 6.3) 9 (21) 12 (29) Ceftriaxone then cefixime 1 17 9 1 (100) 1 (100) Azithromycin 2 7 and 15 3 and 7 0 0 58 patients, with a step-down in 25, 42 and one patient to oral ciprofloxacin, azithromycin or cefixime, respectively. The median duration of antimicrobial therapy varied between 10 and 14 days. The median fever clearance time was 7.7 days with ceftriaxone monotherapy given for a median of 10 days. In the early period of the study, ceftriaxone was followed by a step-down to oral ciprofloxacin for a median of 14 days; the median fever clearance time was 6.4 days. When isolates with intermediate susceptibility to ciprofloxacin were known to be prevalent, the regimen was amended and initial ceftriaxone was followed by a stepdown to oral azithromycin for a median of 13 days. With this regimen the median fever clearance time was 4.4 days, significantly shorter than with ceftriaxone alone (log-rank test p=0.008; Figure 2). We hypothesised that the protracted recovery among children treated with ceftriaxone monotherapy was related to disease severity. The complication rate in children treated with ceftriaxone alone was 38% (22/58), compared with 8% (2/25) among those treated with ceftriaxone followed by ciprofloxacin (p=0.013) and 29% (12/42) in children treated with ceftriaxone followed by azithromycin (p=0.45). When stratified for presence of complicated disease, the fever clearance time remained significantly shorter for the children treated with ceftriaxone followed by azithromycin compared with ceftriaxone alone (logrank p=0.013). A total of 37/128 (29%) and 4/10 (40%) of the hospitalised children with enteric fever and NTS infection developed a complication, respectively (p=0.48). The most common enteric fever complication was gastrointestinal bleeding (Table 4). One child with severe abdominal pain underwent Figure 2. Kaplan Meier plot of the proportion of inpatient children remaining febrile after commencing therapy with: i.v. ceftriaxone throughout treatment for a total median duration of 10 days; or i.v. ceftriaxone followed by a step-down to oral azithromycin for a total median duration of 13 days. a laparotomy, an ileus and swollen gall bladder was found, serovar Typhi was isolated from the gall bladder, but no intestinal perforation was detected. The overall case fatality rate was 2/10 (20%) in children admitted with NTS bacteraemia compared with 2/128 (1.6%) of children admitted Table 4 Complications in Cambodian children admitted to hospital with culture-confirmed enteric fever (n=128) or invasive non-typhoidal salmonellosis (NTS) (n=10) between 2007 and 2011 cases age <5 years (n=36) cases age 5 9 years (n=56) cases age 10 15 years (n=36) All cases with enteric fever (n=128) All cases with NTS (n=10) Complicated disease 10 (28) 20 (36) 7 (19.4) 37 (29) 4 (40) Gastrointestinal bleeding 4 (11) 8 (14) 2 (6) 14 (11) 0 Blood transfusion 1 (3) 6 (11) 3 (8) 10 (8) 0 Jaundice 2 (6) 5 (9) 2 (6) 9 (7) 0 Lung infection 5 (14) 3 (5) 1 (3) 9 (7) 1 (10) Cholecystitis 0 5 (9) 1 (3) 6 (5) 0 Haemodynamic shock 1 (3) 2 (4) 0 3 (2) 2 (20) Encephalopathy 1 (3) 1 (2) 1 (3) 3 (2) 0 Surgery 0 2 (4) 0 2 (2) 0 Relapse 0 1 (2) 1 (2) 1 (1) 1 (10) Death 0 2 (4) 0 2 (2) 2 (20) Data are number (%).

K. Emary et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 106 (2012) 718 724 723 with enteric fever (OR 15.8, 95% CI 1.0 231; p=0.03). A 6- year-old child with enteric fever died within 24 h of admission in septic shock and a second child aged 8 years died after 16 days of admission and ceftriaxone treatment with a large pleural effusion and probable pneumonia. The two children with NTS bacteraemia died within 24 h of admission with septic shock, one was aged 12 years with underlying HIV infection and was one aged 1 month with diarrhoea. Significant factors associated with complicated disease after univariate analysis were hepatomegaly (p<0.001), haemoglobin <10 mg/dl (p=0.014), MDR phenotype (p=0.013) and intermediate susceptibility to ciprofloxacin (p=0.019). After logistic regression for these multiple factors, the presence of hepatomegaly remained independently associated with severe disease (adjusted OR 4.8, 95% CI 3.7 4.9; p=0.004). 4. Discussion We have described a significant burden of antimicrobial-resistant enteric fever in Cambodian children. Serovar Typhi was the commonest isolate from blood cultures in children at this location for the last 5 years and the majority were MDR with intermediate susceptibility to ciprofloxacin. These observations are in keeping with a large community-based study near the capital Phnom Penh and suggest that drug-resistant serovar Typhi is widespread in the country. 10 Our genotyping further demonstrated that the majority of isolates were of the H58 haplotype, occurring concurrently with a gyra mutation inducing a serine to phenylalanine substitution at position 83, a haplotype/phenotype combination, which has become dominant in Asia and some parts of Africa in recent years. 7,13,14 The presence of MDR strains with intermediate susceptibility to ciprofloxacin limits the choice of enteric fever treatment, with ceftriaxone, azithromycin and gatifloxacin as potential options. 15 17 Ceftriaxone was used as the initial empiric therapy for children admitted to hospital as it is suitable for enteric fever and other common invasive bacterial infections. In many cases after the diagnosis was confirmed and the child s condition improved, this was changed to an oral drug. Although antimicrobial resistance to ceftriaxone is rare in invasive Salmonellae, the response to treatment is often slow. 6 The median fever clearance time in this study was 7.7 days with ceftriaxone monotherapy given for 10 days. In some patients a stepdown to oral ciprofloxacin was employed with median fever clearance times of 6.6 days. When it was understood that most strains had intermediate susceptibility to ciprofloxacin, oral ciprofloxacin was substituted with oral azithromycin, and when given for 13 days the median fever clearance time was 4.4 days. Our data, whilst uncontrolled, suggests that in children with fever requiring hospital admission, subsequently confirmed as enteric fever, ceftriaxone followed by a step-down to oral azithromycin is a suitable regimen although the optimum time to stepdown requires further investigation. Gatifloxacin has been shown to be an acceptable alternative in other areas of high DCS prevalence 17 as it is less inhibited by the common mutations of the gyra gene than the older fluoroquinolones, but it is not easily available locally. Significantly, none of the isolates were fully resistant to ciprofloxacin or ceftriaxone which is an emerging problem in some areas. 18,19 This is despite high rates of extended-spectrum -lactamase carriage in other Enterobacteriacae circulating in children in this area. 2 MDR serovar Typhi strains were present in Cambodia in the mid- 1990s (C.M. Parry, personal observation) and appear not to have declined as has been described in other parts of Asia. 5 Molecular genotyping of the strains in this study further demonstrates the dominance of the H58 haplotype with a gyra mutation leading to a S83F amino acid substitution. This strain is also dominant in the Mekong delta in Vietnam and other areas of Asia and is frequently associated with an IncHI1 plasmid carrying the genes for the MDR phenotype, 13,20,21 yet the factors that have led to the success of this particular strain are not yet known. The MICs against ciprofloxacin were in the range expected for isolates with intermediate susceptibility to ciprofloxacin and the values for azithromycin and gatifloxacin were comparable to other studies. 16,17 The reported severity of enteric fever in young children is variable across different studies. 22 Some investigators describe a mild disease with few complications 23 whereas other series describe high rates of complications and mortality, particularly in the very young. 24 26 Anaemia and drug resistance, but not hepatomegaly, have been associated with disease severity in previous studies 24 26 In this study, complications occurred in 28% of children and mortality was 1.6%. The proportion of severe hospitalised cases partly depends on the threshold for hospital admission and the availability of antimicrobials effective against the bacteria causing enteric fever locally. Population-based studies suggest that between 10% and 20% of cases are admitted to hospital with the remainder managed in the community. 6,10 It is also possible that some children from rural Cambodia, with inadequate access to healthcare facilities, may develop severe enteric fever with complications and die without seeking medical attention. We identified a very limited number of cases due to serovar Paratyphi A. This is in contrast to other areas of Asia where the proportion of enteric fever cases due to serovar Paratyphi A is increasing. 27 The factors that determine the relative proportion of serovar Typhi to Paratyphi A in a community are not known, although of note, typhoid vaccination is not widely used in this area. The number of invasive infections with NTS was also low, unlike in some parts of sub-saharan Africa where NTS are a common cause of invasive bacterial infection in children below the age of five years. 28 Most of the children with an invasive NTS infection in this study had previously recognized risk factors namely as a complication of severe diarrhoea in those under 1 year of age or in those with HIV infection. 28,29 It is noteworthy that many of the NTS isolates were resistant to commonly used antimicrobials, as seen in adjacent countries. 4 The control of enteric fever requires the identification of locally important risk factors and includes improving access to clean water, uncontaminated food and the availability of adequate sanitation. 6 The detection and

724 K. Emary et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 106 (2012) 718 724 treatment of chronic carriers has been considered important in controlling enteric fever in developed countries but has not been employed in developing countries because of the difficulty of detecting carriers. Although vaccination as a public health tool is recommended in areas where the burden of enteric fever is high and antimicrobial resistance is a persistent problem, there are few places where it has actually been employed. 30 Additional, prospective community based studies of the epidemiology of enteric fever in this area are required to enable a feasible control strategy to be devised. Authors contributions: KE and CEM contributed equally to this study. CMP, KE, NC, CEM, KC, NES, SB and NPJD conceived and designed the study; all authors participated in the conduct of the study; CMP, KPA, CEM, SS, DPT, TVTN, VW, PA, VK, LW, NES, MJC and SB were involved in the analysis and interpretation of the data; CMP wrote the first draft of the paper. All authors contributed to revising the draft, had full access to all the data and read and approved the final manuscript. CMP and NPJD are guarantors of the study. Acknowledgements: The authors thank the Director of the Angkor Hospital for Children (Cambodia) for his support of this work; the staff of the Microbiology Laboratory and Hospital Records Department for their help conducting the study; and Prof. Sharon Peacock and Sayan Langlah for their contribution in establishing the microbiology laboratory. Funding: The study was funded by the Li Ka Shing- University of Oxford Global Health Program and the Wellcome Trust of Great Britain. The study sponsors had no role in the study design, the collection, analysis, or interpretation of the data, the writing of the report, or the decision to submit the paper for publication. SB is funded by the OAK Foundation through Oxford University. Competing interests: None declared. Ethical approval: The study was approved by the AHC Institutional Review Board and the Oxford Tropical Research Ethics Committee, UK (OXTREC Ref: 45-11). References 1. Coker RJ, Hunter BM, Rudge JW, Liverani M, Hanvoravongchai P. Emerging infectious diseases in southeast Asia: regional challenges to control. Lancet 2011;377:599 609. 2. Emary K, Moore C, Parry C, Khun PA, Ngoun C. Health in Southeast Asia. Lancet 2011;377:1571. 3. Phetsouvanh R, Phongmany S, Soukaloun D, et al. Causes of community-acquired bacteraemia and patterns of antimicrobial resistance in Vientiane, Laos. Am J Trop Med Hyg 2006;75:978 85. 4. Chierakul W, Rajanuwong A, Wuthiekanum V, et al. 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