Invasive Gram-negative bacilli are frequently resistant to standard antibiotics for children admitted to hospital in Kilifi, Kenya

Philip Bejon1,2, Isaiah Mwangi1, Caroline Ngetsa1, Salim Mwarumba1, James A. Berkley1, Brett S. Lowe1, Kathryn Maitland1,3, Kevin Marsh1,2, Mike English1,4 and J. Anthony G. Scott1,2,*

1 Wellcome Trust/Kenya Medical Research Institute (KEMRI), Centre for Geographic Medicine Research — Coast, PO Box 230, Kilifi, Kenya; 2 Nuffield Department of Clinical Medicine, Oxford University, John Radcliffe Hospital, Oxford, UK; 3 Department of Academic Paediatrics, Imperial College, London, UK; 4 Department of Paediatrics, University of Oxford, John Radcliffe Hospital, Oxford, UK


* Corresponding author. Tel/Fax: +254-415-25453/22390; Email: pneumo{at}ikilifi.net

Received 14 February 2005; returned 23 March 2005; revised 25 March 2005; accepted 5 April 2005


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives: To determine the pattern of resistance among Gram-negative bacilli causing invasive bacterial disease for the antibiotics that are already in common use in Kilifi, Kenya and for two potential alternatives, ciprofloxacin and cefotaxime. Also, to determine whether prevalence and severity of resistance was increasing over time, to identify patients who are particularly at risk of resistant infections, and to explore which factors are associated with the development of resistance in our setting.

Methods: We used Etest to study antibiotic susceptibility patterns of 90 Gram-negative bacilli cultured in blood or CSF from paediatric inpatients over 8 years.

Results: Susceptibility to amoxicillin 28%, cefotaxime 95% and ciprofloxacin 99% did not vary significantly with age. Susceptibilities for isolates from children aged less than 14 days were: chloramphenicol, 81%; trimethoprim/sulfamethoxazole, 71%; and gentamicin, 91%. From older children, susceptibilities were: chloramphenicol, 62%; trimethoprim/sulfamethoxazole, 39%; and gentamicin, 73%. Chloramphenicol susceptibility was significantly more common among non-typhi salmonellae than other species (79% versus 53%, P<0.0005). The combination of gentamicin and chloramphenicol covered 91% of all isolates. The prevalence of resistance did not increase over time and was not more common in patients with HIV or malnutrition. Age was the only clinical feature that predicted resistance.

Conclusions: Gentamicin or chloramphenicol alone was suboptimal therapy for Gram-negative sepsis, although in this retrospective study, there was no association between resistance and mortality.

Keywords: antibiotic resistance , Gram-negative infections , developing countries , mortality , children


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Invasive Gram-negative bacilli are associated with high mortality in Kenya and throughout the world.1 Early treatment with an antibiotic to which the organism is susceptible by international standards2 is the mainstay of therapy. In developing countries, blood and cerebrospinal fluid (CSF) cultures are infrequent, reducing the information available to guide treatment at an individual and at a public health level.

In sub-Saharan Africa, the data are patchy. Across five sites in sub-Saharan Africa, resistance to ampicillin and trimethoprim/sulfamethoxazole was almost universal, but susceptibility to chloramphenicol and sometimes gentamicin appeared preserved.39

The objective of this study was to determine the pattern of resistance among Gram-negative bacilli causing invasive bacterial disease for the antibiotics in common use in Kilifi, Kenya and for two potential alternatives; ciprofloxacin and cefotaxime.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Study sample

The study population comprised stored frozen isolates of Gram-negative bacilli cultured from blood or cerebrospinal fluid taken from paediatric inpatients at Kilifi District Hospital (KDH), Kenya, isolated between 1 January 1994 and 31 December 2001. KDH is a rural, government-funded hospital with 42 paediatric beds and an average of 4400 admissions per year. Before August 1998, cultures of CSF and blood were initiated on clinical suspicion of meningitis or sepsis; thereafter all acute admissions, except trauma and burns, were investigated with blood cultures.

Treatment of inpatients at KDH is based on WHO guidelines. Young infants ≤60 days are treated with benzylpenicillin and gentamicin. Older children with suspected meningitis or shock are given benzylpenicillin and chloramphenicol. Amoxicillin or benzylpenicillin is prescribed alone to treat mild or severe pneumonia, respectively; in those with prostration or pulse oximetry readings below 90%, chloramphenicol is substituted. Gentamicin and amoxicillin are used in malnutrition. Trimethoprim/sulfamethoxazole is the commonest outpatient antibiotic in Kilifi District, but is not used for inpatients.

Laboratory methods

Before June 1998, brain heart infusion broth was used to culture blood for 7 days at 37°C in 5% CO2 with sub-cultures at 1, 2 and 7 days. Subsequently, BacTec Peds Plus media (Becton Dickinson, NJ, USA) were incubated for 5 days and sub-cultured as indicated by the BacTec instrument.

Isolates were stored at –70°C in tryptone soya broth with 15% glycerol for up to 8 years. Antimicrobial susceptibilities were determined by Etest in the laboratories of the manufacturer (AB Biodisk, Solna, Sweden).

Data analysis

STATA 8.2 (StataCorp, College Station, TX, USA) was used. Susceptibility breakpoints were taken from the NCCLS guidelines 2003.2 {chi}2 was used to test hypotheses of association. Logistic regression models were developed with backward stepwise regression, excluding variables when the likelihood ratio test P value was ≥0.05. Isolate species and source of isolate were included throughout. Intermediate resistance (I) was classified within resistance (R).


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
During the study period, 801 admissions to the paediatric ward of KDH had Gram-negative bacilli cultured from blood or CSF. In 26 patients, isolates of different Gram-negative bacilli were obtained; these are analysed here as independent episodes. Isolates of Acinetobacter (n=63), non-aeruginosa Pseudomonas (n=43), and other environmental organisms (n=13) were not studied. Of 708 frozen isolates in the target population, 690 were successfully retrieved. Inpatient mortality was 62% (79/128) in children <14 days old and 31% (149/475) in older children.

Table 1 shows the susceptibility patterns of Gram-negative bacilli to four antibiotics that are in frequent use at KDH. Resistance was common, except for two antibiotics that are rarely used in Kilifi: ciprofloxacin and cefotaxime. Non-typhi salmonellae were significantly more susceptible to chloramphenicol than other species (79% versus 53%, P < 0.0005). We calculated the expected cover from combinations of two antibiotics, assuming no synergy or antagonism. Of the commonly available antibiotics, the broadest cover is offered by combining chloramphenicol and gentamicin (91%; 95% CI, 88–93%). Amoxicillin and gentamicin would cover 76% (95% CI, 72–80%), and amoxicillin and chloramphenicol would cover 65% (95% CI, 60–70%).


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Table 1. Resistance patterns of 690 invasive isolates of Gram-negative bacilli for four antibiotics determined by Etest

 
Although resistance to amoxicillin and chloramphenicol was more prevalent before 1997 (Table 2), there was no suggestion of a trend by year of study (data not shown). Susceptibility varied markedly with the age of the patient. Conventional groupings divide very young children at 1 month or 60 days, but variation was best described here by dividing those aged <14 days from older children. For children aged <14 days versus older children, susceptibilities were 93% versus 73% for gentamicin, 81% versus 62% for chloramphenicol, and 71% versus 39% for trimethoprim/sulfamethoxazole (P < 0.0005 for each comparison). Resistance was not associated with the anatomical source of the culture or HIV infection.


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Table 2. Logistic regression odds ratios and 95% CIs in final models of risk factors for resistance to amoxicillin, chloramphenicol, trimethoprim/sulfamethoxazole and gentamicin

 
Significant risk factors for resistance to each of the four commonly used antibiotics, determined by logistic regression, are listed in Table 2. Separate models were specified for each of the four antibiotics, but results are remarkably consistent for chloramphenicol, trimethoprim/sulfamethoxazole and gentamicin. Odds ratios for resistance in the period 1998–2001 compared with 1994–1997 varied from 0.58 to 0.69. Odds ratios for resistance among children ≥14 days old varied from 2.79 to 4.88. Significant associations were observed between a positive malaria slide and isolate susceptibility to amoxicillin, chloramphenicol and trimethoprim/sulfamethoxazole.

Fatal outcome was modelled by logistic regression using 308 patient episodes, including 106 deaths, which had complete data for the explanatory variables HIV status, sex, age and malnutrition. Odds ratios for resistance to individual antibiotics were derived within sub-populations that were likely to have been exposed to the drug according to hospital- and community-based protocols. After excluding neonates (age <28 days), adding terms for trimethoprim/sulfamethoxazole resistance or amoxicillin resistance gave odds ratios of 2.01 (P=0.11) or 0.87 (P=0.75), respectively. For older children without malnutrition (n=132), the odds ratio for chloramphenicol resistance was 2.79 (P=0.15). In malnourished children and neonates, gentamicin resistance was not associated with mortality (OR=1.4, P=0.53, n=214).


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
This study shows that, beyond the first 2 weeks of life, the antibiotics that are used most frequently to treat children with invasive bacteria in Kenya have limited in vitro activity against a representative sample of invasive Gram-negative bacilli. Approximately one-third of these infections are currently treated inappropriately, although for children under 14 days of age, the current practice of treating with benzylpenicillin and gentamicin seems appropriate. Neonates acquire the colonizing bacteria from their mothers, but older infants more often acquire organisms from other children. Since children take antibiotics more often than adults these bacteria experience more antibiotic pressure.

Resistance to amoxicillin, chloramphenicol, trimethoprim/sulfamethoxazole and gentamicin is common, but the situation is not getting worse. The small improvement in susceptibility in the second half of the study period might be explained by sampling all children, including those with milder disease who are less likely to have received antibiotics before presentation. Secondly, the BacTec Peds Plus media used in the latter half might better support growth of antibiotic exposed organisms, increasing the frequency of susceptible isolates. Within the period 1998–2001, there was no trend of increasing resistance. The association between a positive malaria slide and isolate susceptibility might reflect prior use of antibiotics and antimalarials before presentation, simultaneously clearing parasites and selecting resistant bacteria.

A significant association was not found between antibiotic resistance and mortality. However, the study was retrospective with reduced numbers after dividing children into groups receiving indicated antibiotics and lacked individual chart review.

Targeting therapy at Gram-negative infections is largely theoretical in the developing world without culture diagnosis, where clinical features determine treatment based on syndromic classification. Analysis of the same inpatient data by syndrome suggests that coverage of WHO recommended antibiotics is in the range 76–97%, owing to the preponderance of Gram-positive organisms. A notable exception is malnutrition; where 65% of bacteraemic isolates are Gram-negative.

Alternatives for antibiotic treatment of Gram-negative sepsis in older children include combination therapy with chloramphenicol and gentamicin, or monotherapy with ciprofloxacin or cefotaxime. In vitro studies and animal models show potential antagonism between gentamicin and chloramphenicol,10 but clinical data suggesting worse outcomes from this or similar antibiotic interactions are not available. More broadly effective antibiotics would increase per-patient costs for a 15 kg child treated parenterally for 7 days from 38 cents and $2.30 for gentamicin and chloramphenicol to $108 or $73 for cefotaxime or ciprofloxacin (2 days parenteral, then oral therapy).

Ultimately, therapeutic decisions in any region should not be determined by data from a single site, yet in Africa there is no functioning mechanism to aggregate data from several sites and determine a regional response. Continent-wide networks, such as the WHO network for antimicrobial surveillance (WHONET) and the WHO paediatric bacterial meningitis surveillance project (WHO-AFRO PBM) provide a forum and limited funding for sentinel surveillance but considerable investment in the culture and infrastructure of antimicrobial surveillance, data aggregation and policy response is required. Finally, given the uncertain predictive value of in vitro testing, therapeutic recommendations requiring increased costs would be best guided by prospective clinical trials.


    Acknowledgements
 
This study is published with the permission of the director of the Kenya Medical Research Institute, Nairobi. The study was supported by KEMRI and by the Wellcome Trust of Great Britain. P. B. holds a Wellcome Trust training fellowship (073597), J. A. B. held a Wellcome Trust training fellowship (053439), J. A. G. S. (061089) and M. E. (050563) hold Wellcome Trust career development fellowships and K. Marsh holds a Wellcome Trust senior fellowship (061702). None of us has any conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
1. Berkley JA, Lowe BS, Mwangi IS et al. Bacteremia among children admitted to a rural hospital in Kenya. N Engl J Med 2005; 352: 39–47.[Abstract/Free Full Text]

2. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests: Approved Standard M2-A8. Supplemental Tables M100-S13. NCCLS, Wayne, PA, USA, 2003.

3. Omari MA, Malonza IM, Bwayo JJ et al. Pattern of bacterial infections and antimicrobial susceptibility at the Kenyatta National Hospital. Nairobi, Kenya. East Afr Med J 1997; 74: 134–7.

4. Malonza IM, Omari MA, Bwayo JJ et al. Community acquired bacterial infections and their antimicrobial susceptibility in Nairobi. Kenya. East Afr Med J 1997; 74: 166–70.

5. Musoke RN, Revathi G. Emergence of multidrug-resistant gram-negative organisms in a neonatal unit and the therapeutic implications. J Trop Pediatr 2000; 46: 86–91.[Abstract/Free Full Text]

6. Walsh AL, Phiri AJ, Graham SM et al. Bacteremia in febrile Malawian children: clinical and microbiologic features. Pediatr Infect Dis J 2000; 19: 312–8.[CrossRef][ISI][Medline]

7. Mokuolu AO, Jiya N, Adesiyun OO. Neonatal septicaemia in Ilorin: bacterial pathogens and antibiotic sensitivity pattern. Afr J Med Sci 2002; 31: 127–30.

8. Ohene A. Bacterial pathogens and their antimicrobial susceptibility in Kumasi, Ghana. East Afr Med J 1997; 74: 450–5.

9. Tenssaie ZW. Multiple antimicrobial resistance in gram negative bacilli isolated from clinical specimens. Jimma Hospital, southwest Ethiopia. Ethiop Med J 2001; 39: 305–12.

10. Sanderson PJ. Gentamicin and chloramphenicol in neonatal meningitis. Lancet; 1978; ii: 210.





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