Systematic Review of Antibiotic Resistance Rates Among Gram-Negative Bacteria in Children With Sepsis in Resource-Limited Countries

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1 Original Article Systematic Review of Antibiotic Resistance Rates Among Gram-Negative Bacteria in Children With Sepsis in Resource-Limited Countries Kirsty Le Doare, 1,2 Julia Bielicki, 2 Paul T. Heath, 2 and Mike Sharland 2 1 Wellcome Centre for Global Health Research, Imperial College, London; and 2 Paediatric Infectious Diseases Research Group, St George s University of London, UK Corresponding Author: Kirsty Le Doare, MRCPCH Paediatric Infectious Diseases Research Group, St George s University of London, Cranmer Terrace, London SW17 0RE, UK. kirsty.ledoare@nhs.net. Received October 9, 2013; accepted January 16, 2014; electronically published March 24, Background. Gram-negative antimicrobial resistance (AMR) is of global concern, yet there are few reports from low- and low-middle-income countries, where antimicrobial choices are often limited. Methods. This study offers a systematic review of PubMed, Embase, and World Health Organization (WHO) regional databases of Gram-negative bacteremia in children in low- and low-middle-income countries reporting AMR since Results. Data included 30 studies comprising children, of whom 7056 had positive blood cultures, and Gram-negative organisms were isolated in 4710 (66.8%). In neonates, Klebsiella pneumoniae median resistance to ampicillin was 94% and cephalosporins 84% in Asia; 100% and 50% in Africa. Large regional variations in resistance rates to commonly prescribed antibiotics for Salmonella spp. were identified. Multidrug resistance (resistance to ampicillin, chloramphenicol, and cotrimoxazole) was present in 30% (interquartile range [IQR], ) in Asia and 75% (IQR, ) in Africa. Conclusions. There is a need for an international pediatric antimicrobial resistance surveillance system that collects local epidemiological data to improve the evidence base for the WHO guidance for childhood Gramnegative bacteremia. Key words. antimicrobial resistance; children; epidemiology; Gram-negative bacteremia. Bacteremia remains a leading cause of death in children under 5 years of age worldwide with an estimated 6% of neonatal and 14% of childhood deaths, although there are few robust estimates of the burden of bacteremia in children in resource-poor settings [1]. The treatment of childhood bacteremia is threatened by the steady increase in the prevalence of antimicrobial resistance (AMR), especially in low- and low-middle-income countries where the availability of antibiotics and cost of therapy are critical constraints. The prevalence of AMR in resource-poor countries has been reviewed previously [2 4]. However, many of the studies discussed earlier represent data at least a decade old, and AMR rates are known to be rising rapidly. In many cases, published data are not amenable to accurate quantitative assessment, particularly in countries whose systematic surveillance systems are basic or absent. The World Health Organization (WHO) has made recommendations to tackle the threat of global AMR [5], the theme for World Health Day 2011, and subsequently published a policy package [6]. Diagnostic laboratory facilities for the evaluation of AMR are frequently limited due to cost, infrastructure, and personnel constraints in lowand low-middle-income settings. In addition, the limited surveillance data available are often based on laboratory analysis without accurate clinical details or timing of blood cultures; therefore, accurate assessments of rates of community-acquired AMR are not possible [7]. Resistance among Gram-negative bacteria (GNB) is now the key global concern, because the spread of multidrugresistant (MDR) GNB is increasing and there are a very limited number of antimicrobial agents in late-stage development [8]. Gram-negative bacteria, particularly non-typhoidal Salmonella, now equal or exceed Gram-positive organisms in importance in several published reports on bloodstream infections (BSIs) in children from African countries [3, 9 11] and represent around half of infections in published studies Journal of the Pediatric Infectious Diseases Society, Vol. 4, No. 1, pp , DOI: /jpids/piu014 The Author Published by Oxford University Press on behalf of the Pediatric Infectious Diseases Society. All rights reserved. For Permissions, please journals.permissions@oup.com.

2 12 Le Doare et al from Asia [4]. Most reports concentrate on adult data without differentiating AMR rates in children. The WHO Pocket Book of Hospital Care for Children offers guidance on clinical diagnosis and management of suspected bacterial infections in children in resource-poor settings [12]. The generic recommendations list only 8 major antibiotics (Table 1) for the treatment of all severe bacterial infections in neonates and children. We conducted a systematic review of studies that used blood culture to identify AMR rates in GNB-BSIs among children from low- and low-middle-income countries with predefined inclusion criteria, published after 2001 when the WHO strategy for containment of AMR was launched. METHODS We conducted a systematic review of the literature between 2002 and 2013 that was in accordance with PRISMA requirements [13]. Search Methods to Identify Studies We searched 2 major scientific databases (MEDLINE and Embase) and the African, Asian, and Latin American sections of the WHO regional databases, using criteria previously reported [14, 15]. MEDLINE and Embase were searched with the search string shown in Appendix 1. String searches for the WHO African, Asian, and Latin American regional databases are also described in Appendix 1. In summary, the subject headings and keywords used were as follows: pediatrics OR child OR infant OR neonate (medical subject heading [MeSH] terms) AND (infection OR sepsis OR bacteremia [MeSH terms] OR fever OR drug resistance, bacterial [MeSH terms] OR AMR). The abstracts and titles from all years and in all languages were compiled in EndNote (Thomson Reuters) and reviewed by 2 authors. Selection of Studies Each article identified by this process as a candidate for inclusion was retrieved as full text. All references from Table 1. Antibiotic Generic Recommendations From the WHO Pocket Book of Hospital Care for Children, Second Edition [12] Condition Drug Dose Dysentery Ciprofloxacin oral Second line: Ceftriaxone IV 15 mg/kg BD for 3 days mg/kg daily for 3 days Mastoiditis Cloxacillin/Flucloxacillin Second line: Ceftriaxone IV 50 mg/kg QDS for 10 days Meningitis 1. Ceftriaxone IV or 2. Cefotaxime IV or 3. If no known resistance locally to chloramphenicol and β-lactams: Chloramphenicol IV plus Ampicillin or Chloramphenicol plus Benzylpenicillin IV 50 mg/kg for 7 10 days 50 mg/kg QDS for 7 10 days 25 mg/kg QDS for 10 days 50 mg/kg QDS for 10 days 60 mg/kg ( U/kg) QDS for 10 days Osteomyelitis Cloxacillin/Flucloxacillin/Ceftriaxone IV if > 3 yrs 50 mg/kg QDS for 10 days Otitis Media, Acute Amoxicillin oral or where no known resistance to co-trimoxazole then give cotrimoxazole 40 mg/kg BD for 5 days 4 mg/kg plus 20 mg/kg BD for 5 days Infant Sepsis and Meningitis Older Child Sepsis Typhoid Urinary Tract Infection Pneumonia Ampicillin IV plus Gentamicin IV or Ceftriaxone IV If staphylococcal infection is suspected Flucloxacillin IV plus Gentamicin Ampicillin IV plus Gentamicin IV or Ceftriaxone IV If Staphylococcal infection is suspected Flucloxacillin IV plus Gentamicin Chloramphenicol Second line: Add ampicillin Co-trimoxazole oral Second line: ampicillin plus gentamicin Ampicillin IV plus Gentamicin IV or Ceftriaxone IV If staphylococcal infection is suspected Cloxacillin IV plus Gentamicin 50 mg/kg QDS 7 10 days (3 weeks for meningitis) mg/kg daily 7 10 days (3 weeks for meningitis) 50 mg/kg daily 7 10 days (3 weeks for meningitis) 50 mg/kg QDS 7 10 days 7.5 mg/kg daily 7 10 days 50 mg/kg daily 7 10 days 50 mg/kg QDS for 7 10 days 25 mg/kg TDS 14 days 50 mg/kg daily QDS 4 mg/kg plus 20 mg/kg BD for 5 days 50 mg/kg QDS 7 10 days 7.5 mg/kg daily 7 10 days 80 mg/kg daily 7 10 days 50 mg/kg QDS for 7 10 days Abbreviations: BD, 2 times a day; IV, intravenous; TDS, 3 times a day; QDS, 4 times a day; WHO, World Health Organization.

3 AMR in Gram-Negative Childhood Infections 13 included articles were also screened for inclusion. When we found duplicate reports of the same study in preliminary abstracts and articles, we analyzed data from the most complete data set. The last search date was December 14, Included studies were required to have identified a recognized standard for the interpretation of antibiotic susceptibility testing (eg, Clinical and Laboratory Standards Institute (CLSI); EUCAST; British Society for Antimicro-bial Chemotherapy) and to have used a recognized method to measure the effect of the antimicrobial agent on the bacteria grown in culture. Furthermore, all included studies had to define how cases of presumed sepsis were identified. All articles were required to quantify the total number of blood cultures obtained, the total number of pathogenic isolates, and to detail the numbers of patients from whom these pathogens were isolated. Only studies meeting our minimum requirements for data completeness described above were included. During abstract review, we excluded review articles, editorials, policy statements, and research exclusively in children who were immunocompromised (ie, from human immunodeficiency virus [HIV] infection). We also excluded conference abstracts, articles describing illness in people living in high-middle and high-income countries (defined by World Bank criteria [16]), case reports, and studies during an epidemic or outbreak. During full-text review, we excluded studies of mixed adult and child populations in which data were not reported separately, those with data collection commencing prior to 2002, and those that used subjective or poorly defined inclusion criteria. Articles that reported patient cohorts already included in other studies were excluded. Data Extraction Data were extracted by the main author using a Microsoft Excel 2007 spreadsheet designed for this review; this information included hospital setting and location, country, study time frame, specific inclusion or exclusion criteria, and culture techniques. Quantitative data collected included number of patients, age range, pathogens isolated, antimicrobial susceptibilities, and use of additional tests (eg, for HIV or malaria). After extraction, data were reviewed and compared by the second author. Instances of disagreement between the 2 extractors were solved by a consensus among the investigators. Whenever needed, we obtained additional information about a specific study by directly contacting the principal investigator. Validity Assessment We used the Grading of Recommendations Assessment, Development and Evaluation approach to summarize the quality of evidence for each outcome [17]. Study type, quality, limitations, imprecision, inconsistency, indirectness, or suspicion of a reporting bias are assessed and graded. A final score based on these categories was then used to grade evidence into 4 categories: (1) high, (2) moderate, (3) low, or (4) very low quality. The grade of evidence for each study is shown in Supplementary Data Table A and B. Definitions Neonatal studies were defined as those including only, infants less than 1 month of age. Pediatric studies were defined as those in which all included patients were aged 0 16 years; data from children aged 0 16 years in studies with mixed populations of both adults and children were extracted and reanalyzed. Antimicrobial resistance was defined as resistance of an isolated pathogen to the antibiotic in question using a standardized antimicrobial susceptibility-testing model such as the agar diffusion test (the Kirby-Bauer method) or other standardized methods for determining the zone inhibition or minimum inhibitory concentration (MIC) of the isolate. Statistical Analysis We calculated crude proportions and ranges of individual pathogens resistant to ampicillin, gentamicin, chloramphenicol, ciprofloxacin, cotrimoxazole, and ceftriaxone in infants and children in Asia and Africa. These proportions were then weighted according to study sample size. These weighted proportions were multiplied by the weighted proportions of all cases of sepsis caused by that pathogen. The resulting weighted median and interquartile ranges (IQRs) attributable to a pathogen that is resistant to recommended antibiotics are then reported. RESULTS Characteristics of the Studies The online database search performed on December 14, 2013 yielded 4922 articles (Figure 1): 3721 were unique to PubMed, 1002 unique to Embase, 191 unique to WHO regional databases, and 8 conference abstracts. Most of these articles were excluded on the basis that research was not conducted in a low or low-middle income country, had data collection commencing prior to 2002, reported results for adults only, or reported results that were not from blood cultures. Three hundred ninety full-text articles (17 in French, 12 in Portuguese, 7 in Spanish, 4 in Chinese, 350 in English) were obtained for more detailed evaluation. Three hundred sixty articles were excluded during detailed screening for not containing data on AMR in blood cultures. Thirty selected articles, representing unique patient cohorts, were eligible for systematic review. The 30 eligible studies were undertaken in 17 locations. Fifteen came from Asia and South East Asia: Pakistan (n = 1) [18], Vietnam (n = 1) [18], India (n = 7) [19 25], Bangladesh (n = 2) [26, 27], Nepal (n = 2) [28, 29], Malaysia (n = 1) [30], and

4 14 Le Doare et al Figure 1. Flowchart of study selection process (based on PRISMA flowchart). AMR, antimicrobial resistance; CSF, cerebrospinal fluid; HIV, human immunodeficiency virus; WHO, World Health Organization. Cambodia (n = 2) [31, 32]. Fifteen came from Africa and the Middle-East: Gambia (n = 1) [33], Ghana (n = 2) [34, 35], Uganda (n = 1) [36], Egypt (n = 1) [47], Palestine (n = 1) [37], Nigeria (n = 3)[38 40], Tanzania (n = 2) [41, 42], Mozambique (n = 1) [43], Mali (n = 1) [44], and Congo (n = 2) [45, 46]; see Figure 2. There were no studies of children reporting AMR in Gram-negative infections since 2001 from South America (Supplementary Data).

5 AMR in Gram-Negative Childhood Infections 15 Figure 2. Overview of study location by continent. (A) Location of Asian studies; (B) Location of African studies. For color, see the figure online. The articles included in this review describe analyses undertaken between 2002 and 2010, in infants and children: from Asia and South East Asia and from Africa and the Middle East. Patients (49 452; 69.3%) were exclusively from pediatric cohorts. Twenty-three studies reported results from communitybased surveillance [18 21, 23 29, 31 37, 41 44, 46], three studies reported hospital-acquired infections [30,

6 16 Le Doare et al 37, 47], whereas the remaining four reported laboratorybased studies of children with febrile illness, where we were unable to determine length of hospital stay prior to blood culture. Basic criteria for inclusion were clinical signs of sepsis, WHO criteria for severe sepsis, and pneumonia. All studies described the microbiological techniques used, although culture media and methods of identification of organisms varied between studies. Blood cultures were positive in 7056 patients across all studies (13.9% of all blood cultures taken), and GNB were isolated in 4710 (7.7% of all cultures taken; range, 4.2% 100%). Gram-negative bacteria accounted for 66.8% of all isolates in these studies. The median age of children with GNB in African studies was 15 months (IQR, 0 months to 5 years), although most populations included all ages from 0 to 15 years, whereas the median age of children in Asian studies was 2 years (IQR, 3 months to 5 years). Pathogen and AMR Rates in Neonates Eleven studies reported results from neonatal units: three in Africa [39, 41, 42], one in the Middle East [37], one in South East Asia [30], and six in Asia [20 22, 25, 26, 28] (Supplementary Data Table A and B). In all areas, Klebsiella pneumoniae was the predominant pathogen, accounting for 49.8% of all GNB, followed by other Enterobacteriaceae. Salmonella species was an important organism in Asian studies but was not reported in any African neonatal study. Pathogen-Specific Resistance. Klebsiella spp. demonstrated the highest level of resistance overall---with resistance to ampicillin 93.8% (IQR, ) and gentamicin 68.8% (IQR, ) reported in Asian studies and 100% (IQR, 0 100) and 54.2% (IQR, ) in African studies. Ceftriaxone resistance was also high: 84.4% (IQR, ) in Asia and 50.0% (IQR, ) in Africa. Enterobacteriaceae demonstrated similar trends with resistance to ampicillin 79.6% (IQR, ), gentamicin 22.2% (IQR, ), and ceftriaxone 74.1% (IQR, 0 100) in Asian studies. Numbers of Escherichia coli (E. coli) were too small to draw meaningful conclusions in Asia, but in Africa studies also demonstrated high or moderate levels of resistance to ampicillin and gentamicin (92.9% [IQR, 0 100] and 42.9% [IQR, ], respectively) (Table 2). Community- and Hospital-Acquired Infections. Hospitalacquired neonatal infections were reported in two studies [30, 37], where Klebsiella, Acinetobacter, and Pseudo-monas were identified as the three major organisms, respectively. These organisms demonstrated 61.1% 85.6% resistance to ceftriaxone and cefotaxime, moderate resistance to piperacillin-tazobactam (18.6%, 11.0%, 9.5%), and low resistance to imipenem (0.4%, 4.2%, 0%). Basis of Resistance. Four studies reported rates of extended spectrum β-lactamase (ESBL)-producing Klebsiella spp. One study from Tanzania reported 49% of Klebsiella as ESBL-producing [41]. In two reports from India, rates of ESBL-producing Klebsiella ranged from 48% to 58% of samples [21, 22]. The highest rates of MDR Klebsiella and E. coli were 82% in a small Indian neonatal study [25]. Pathogen and AMR Rates in Children Key Pathogens Identified. Salmonella spp., especially nontyphoidal Salmonella, were the most common GNB reported in children in Asia. The isolates demonstrated over 32% resistance to ampicillin and 10% resistance to ciprofloxacin [18 20, 23, 24, 29, 34]. Over one third of isolates (37.3%) were resistant to nalidixic acid [18, 24, 29]. Higher rates were found in African studies (ampicillin 85.0% and ciprofloxacin 10%, respectively) [33, 34, 36, 45]. Table 2. Median Percentage Antimicrobial Resistance of Gram-Negative Bacteria in Neonates in Low and Low-Middle Income Countries Pathogen N Ampicillin Median % Resistance (IQR) Gentamicin Median % Resistance (IQR) Chloramphenicol Median % Resistance (IQR) Cotrimoxazole Median % Resistance (IQR) Ciprofloxacin Median % Resistance (IQR) Ceftriaxone Median % Resistance (IQR) Asia E. coli ( ) 83.3 ( ) 0 (0 43.8) 31.3 (0 90.6) 4.5 (0 44.5) 80.2 ( ) Klebsiella spp ( ) 68.8 ( ) 41.9 ( ) 16.7 (0 55.2) 32.5 ( ) 84.4 ( ) Enterobacter ( ) 22.2 (0 70.4) 0 (0 44.4) 0 (0 63.0) 5.6 (0 47.2) 74.1 (0 100) spp. Acinetobacter 93 0 (0 51.7) 38.7 (0 86.7) 0 (0 43.3) 0 (0 35.0) 10.3 (0 43.3) 61.1 ( ) spp. Pseudomonas (0 57.5) 19.4 (0 38.1) 0 (0 32.2) 0 (0 57.1) 5.6 (0 24.2) 53.8 ( ) spp. Africa E. coli (0 100) 42.9 (0 68.2) NR 0 (0 77.3) 0 (0 4.5) 0 (0 50.0) Klebsiella spp (100) 54.5 (0 68.0) NR 0 (0 80.0) 0 (0 10.0) 50.0 (0 86.5) Abbreviations: IQR, interquartile range; NR, not recorded.

7 AMR in Gram-Negative Childhood Infections 17 Table 3. Median Percentage Antimicrobial Resistance of Gram-Negative Bacteria in Children in Low and Low-Middle Income Countries Pathogen Pathogen-Specific Resistance. Resistance of other Enterobacteriaceae, including E. coli and Klebsiella, were widely reported in African studies. High levels of resistance to ampicillin 58.0% (0% 100%), gentamicin 30.0% (0% 51.2%), and ceftriaxone 30% (0% 64%) were reported in Klebsiella infections, and over 50% resistance rates to ampicillin were noted for E. coli isolates [33, 36, 38, 40](Table3). Minimum Inhibitory Concentrations. Few studies report MIC to any of the antibiotics reported, which makes it difficult to assess the intermediate or decreased susceptibility of different pathogens to the antibiotics tested. Increased MICs were reported in five studies [24, 27, 32, 45, 6]. These studies mostly focused on increased MIC to ciprofloxacin ranging from MIC mg/l to > 3 mg/l in Asia and South East Asia [24, 27, 32] and mg/l to 0.25 mg/l in Africa [45, 46]. One study from the Congo reported increased MICs (MIC 90 )to Salmonella spp. of 16 and 4 mg/l for nalidixic acid and ciprofloxacin, respectively [45]. Hospital-Acquired Infection. Zaki Mel [47] reported high rates of resistance to ceftriaxone (65%) in hospitalacquired Klebsiella infection in a pediatric population in Egypt (0 15 years), 50% resistance to amikacin, and 30% resistance to imipenem. This study found 42% of Klebsiella spp to be ESBL producers. DISCUSSION N Ampicillin Median (IQR) Gentamicin Median (IQR) In low- and low-middle-income countries, the high burden of BSI in young children together with the limitations in diagnostics make it essential that effective and evidencebased empiric antibiotic guidelines and therapy are available. The WHO clinical guidelines for the management of common childhood illnesses take into account the constraints of resource-poor settings. For children with signs of BSI but no localizing signs such as pneumonia or meningitis, empiric antibiotic therapy aims to broadly cover the most likely causes of BSI for the relevant age group. Chloram phenicol Median (IQR) Cotrimoxazole Median (IQR) Ciprofloxacin Median (IQR) Ceftriaxone Median (IQR) Asia E. coli ( ) 83.3 ( ) 0 (0 21.9) 0 (0 71.9) 0 (0 20.9) 90.6 ( ) Klebsiella spp (0 100) 68.8 ( ) 0 (0 41.9) 0 (0 47.9) 20.0 ( ) 66.7 ( ) Salmonella spp (0 49.1) 4.1 (0 54.1) 19.7 (1 59.6) 30.0 ( ) 0 (0 18.3) 0 (0 9.8) Africa E. coli ( ) 11.1 (0 20.8) 33.3 (0 87.2) 50.5 (0 77.8) 0 (0 10.9) 0 (0 10.9) Klebsiella spp (0 100) 30.0 (0 51.0) 0 (0 58.0) 80.0 (0 100) NR 30 (0 64.0) Salmonella spp ( ) 0 (0 27.1) 72.3 ( ) 75.6 ( ) 0 (0 3.8) NR H. influenzae (0 100) 20.1 ( ) 24.2 ( ) 81.0 ( ) NR 6.1 (0 25.6) Abbreviations: IQR, interquartile range; NR, not reported. However, AMR varies both within and between countries, and local as well as national data are required to effectively manage GNB. This review identified that, in Asia, very high levels of resistance to ampicillin and gentamicin are present in neonatal GN BSI. Although AMR rates are lower in African studies, approximately one half of isolates in infants and young children were resistant to ampicillin and gentamicin. Antimicrobial resistance was highest in isolates of K. pneumoniae in both Africa and Asia, similar to the findings of Downie et al [48] in their recent review of communityacquired neonatal sepsis. Recent evidence from Africa highlights the importance of healthcare-associated infection in children [49]. We also found high levels of GNB AMR in neonatal units. Although ceftriaxone is considered to offer adequate coverage for Gram-negative organisms in older children, our findings suggest increasing AMR rates, especially in Asia, now threaten its efficacy. This result could account, at least in part, for the widespread exposure to antibiotics in the community, which may drive AMR [50]. It is clear that continued efforts to prevent both community-acquired and healthcare-associated infections in low- and middle-income settings are required. The finding of regional differences in susceptibility of Salmonella spp. to the WHO first-line treatment recommendations (chloramphenicol, ampicillin, and cotrimoxazole) is concerning. We found approximately 75% of isolates in Africa to be MDR (definedasresistancetoampicillin,cotrimoxazole, and chloramphenicol) and 30% of Asian and South East Asian isolates to be MDR. Nontyphoid salmonella resistance is thought to be due, in part, to inch1 plasmid, which has been found in outbreaks in India, Pakistan, and Kenya [51]. However, susceptible strains seem to be reemerging in Asia [52]. In our data, low rates of resistance to ampicillin, chloramphenicol, and cotrimoxazole were identified in Cambodia, Nepal, and Vietnam, and higher rates of resistance were found in India and Pakistan. This finding is in agreement with other papers that suggest that the inchi1

8 18 Le Doare et al plasmid is mostly absent in Nepal, Vietnam, and Cambodia [29]. Adult data also highlight regional differences in antimicrobial susceptibility of different Salmonella serotypes. The most recent surveillance of 14 countries (including only two studies from Asia and Africa) suggests that up to 100% of isolates are MDR in Uganda compared to 67% of isolates in the Philippines [53]. Our review has several important limitations including representativeness and heterogeneity. Hospital type, population characteristics, enrollment criteria, and study period varied widely, which raises concerns about the appropriateness of our aggregation of the different datasets. Although this review includes data from more than 7000 episodes of bacteremia from 17 countries, this sample is a very small proportion of the annual global burden of bacterial infections. However, in the absence of other sources of information, we consider that our method is one way to help understand the rapidly evolving patterns of AMR in low and low-middle income countries. Clinical outcome data are vital to interpret the relationship between reported rates of AMR and empiric-prescribing regimens. Approximately half of the studies in this review were laboratory-based, so the accurate identification of community-acquired infection was problematic. The use of laboratory-based studies, in particular, could overestimate Gram-negative AMR rates because these children are likely to be the sickest patients, who have often failed empiric antibiotic regimens in the community and may be more likely to have an AMR pathogen. Data were mainly from large tertiary centers in urban areas, making it difficult to be certain that AMR rates are representative of rural communities, with potentially even less access to second-line antimicrobial agents. In addition, the quality of the studies varied greatly, and few high-quality studies were available. This variation in study quality will have had an impact on the weighted proportions we report for AMR in the studies. Few of the reported studies identified local guidelines for the treatment of GNB, and only one third identified the use of the WHO guidelines for treatment of pediatric BSI. Use of a standardized reference method such as CLSI/National Committee for Clinical Laboratory Standards is important to ensure that susceptibility results are reproducible and accurate. When antibiotic choices are made, it is often assumed that in vivo efficacy is closely related to in vitro susceptibility. When a reference method is not used, it is difficult to be certain of the correlation between in vitro results and in vivo efficacy. The efficacy of these antimicrobials may have been underestimated due to missing data. In addition, antibiotic pretreatment is a major confounder in many areas of Asia and Africa, and reporting of prior antibiotics was variable. None of the studies from Asia and South East Asia and only three studies from Africa reported rates of prior antimicrobial use. Although selection and development of AMR is known to be related to both overuse and misuse (underuse, overuse, and incomplete or prolonged treatment), these factors need to be considered when considering these data. We have excluded studies that only contained children with HIV infection, which may affect the generalizability of our results. These children were excluded because although HIV and acquired immunodeficiency syndrome primarily affects children in developing countries and is an important risk factor for invasive bacterial disease, the scope of this review was to assess AMR in otherwise healthy children, for whom the WHO recommendations are primarily developed. The findings in this review have significant implications for WHO empiric antibiotic recommendations. For neonates, sepsis due to resistant GNB is an emerging and substantial problem, and the currently recommended first-line ( penicillin/ampicillin plus gentamicin) or second-line antibiotics (ceftriaxone) seem to provide less than optimal cover in a significant proportion of cases. For children, the use of ampicillin, gentamicin, chloramphenicol, cotrimoxazole, and ceftriaxone for bacteremia other than salmonella also provide less than optimal coverage in some settings for both bacterial meningitis and septicemia. Appropriate second-line treatment when GNB are isolated or suspected need to be explored, and clinical indications for timely second-line therapy need to be considered. At the same time, we need to better understand the epidemiology of invasive bacterial infections in this setting. This comprehension should include investigation of how best to support widespread use of detailed microbiological evaluation of appropriate samples in this setting. There is an urgent need to support antimicrobial stewardship programs and rational antibiotic use in both the community and the hospital settings globally in order to minimize antibiotic pressures and preserve the few current antimicrobials that are effective in the treatment of GNB in children. Infection control measures, especially in the hospital setting, should be advocated to prevent the further spread of drug-resistant organisms. CONCLUSIONS There remains a clear policy gap between evidencebased empiric guidelines and prospective, regional clinicalepidemiological surveillance to monitor the changing patterns of AMR within and between WHO regions. The Transatlantic Taskforce on Antimicrobial Resistance has highlighted standardized surveillance and prevention

9 AMR in Gram-Negative Childhood Infections 19 strategies to improve comparisons in data between Europe and the United States, and the WHO taskforce on AMR has highlighted the need for regional surveillance measures to ensure better use of antimicrobials and innovative approaches to prevent infection, including vaccination programs, as key priorities for children s health [54]. Our review highlights the paucity of international pediatric AMR surveillance data in low- and low-middleincome countries since It is interesting to note that the majority of countries reporting data included in this analysis have recently published policy documents for AMR, but this has yet to be translated into specific pediatric AMR-focused surveillance programs. Surveillance has been successful in monitoring resistance in malaria [55] and MDR tuberculosis [56, 57]. There are no similar global surveillance studies for AMR as yet, although some regional programs are well established. Funding: KLD is funded by a Wellcome Trust Global Health Fellowship. Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Supplementary Data Supplementary materials are available at the Journal of the Pediatric Infectious Diseases Society online ( oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author. References 1. Black RE, Cousens S, Johnson HL, et al. Global, regional, and national causes of child mortality in 2008: a systematic analysis. Lancet 2010; 375: Vlieghe E, Phoba MF, Tamfun JJ, Jacobs J. 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