J Med Microbiol 56 (2007), 1479-1484; DOI: 10.1099/jmm.0.47416-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Molecular epidemiology of community-acquired invasive non-typhoidal Salmonella among children aged 2–29 months in rural Gambia and discovery of a new serovar, Salmonella enterica Dingiri
Usman N. Ikumapayi1,
Martin Antonio1,
Jacob Sonne-Hansen2,
Ekow Biney1,
Godwin Enwere1,
Brown Okoko1,
Claire Oluwalana1,
Adeola Vaughan1,
Syed M. A. Zaman1,
Brian M. Greenwood3,
Felicity T. Cutts1 and
Richard A. Adegbola1
1 Medical Research Council Laboratories, Banjul, The Gambia, West Africa
2 Danish Salmonella Centre, Statens Serum Institute, Copenhagen, Denmark
3 London School of Hygiene and Tropical Medicine, Keppel Street, London, UK
Correspondence
Martin Antonio
mantonio{at}mrc.gm
Received 26 May 2007
Accepted 23 July 2007
Sixty-two invasive non-typhoidal Salmonella (NTS) isolates from children aged 2–29 months in rural Gambia were examined for serovar prevalence and antimicrobial susceptibility, and characterized using multilocus sequence typing (MLST) of seven genes, aroC, dnaN, hemD, hisD, purE, sucA and thrA. Salmonella enterica serovar Enteritidis was the most common serovar (80.6 %), followed by S. enterica serovar Typhimurium (8.0 %). Thirty-three per cent of the isolates were resistant to all eight antimicrobials tested, including ampicillin (74.2 %), cotrimoxazole (64.5 %) and tetracycline (63 %). A total of 40.3 % of the NTS cases had an initial clinical diagnosis of malaria, whilst 27.3 % had a diagnosis of clinical pneumonia and 18 % had a diagnosis of septicaemia. MLST of NTS resulted in ten different sequence types (STs), of which five were novel, representing five different NTS serovars. In general, STs were restricted to the same serovar. One type (ST11) encompassed 80.6 % of the NTSs. A new NTS serovar named S. enterica serovar Dingiri was discovered. S. Dingiri was isolated from a 6-month-old male with an initial clinical diagnosis of malaria but a final clinical diagnosis of anaemia and septicaemia. S. Dingiri, which possesses an antigenic formula of 17:z:1,6, was sensitive to ampicillin, cefotaxime, chloramphenicol, ciprofloxacin, cotrimoxazole and tetracycline but resistant to gentamicin, and was ST338.
Abbreviations: HIV, human immunodeficiency virus; MLST, multilocus sequence typing; NTS, non-typhoidal Salmonella; PVT, pneumococcal-conjugate vaccine trial; ST, sequence type.
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INTRODUCTION
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Non-typhoidal Salmonella (NTS) is one of the most important enteric pathogens causing bacteraemia in young children in many parts of the world, including Africa (Hill et al., 2007; Enwere et al., 2006; Berkley et al., 2005; Mulholland & Adegbola, 2005). Extraintestinal infections caused by Salmonella species are a common cause of severe morbidity and mortality among young children from tropical Africa (Hill et al., 2007; Enwere et al., 2006; Berkley et al., 2005). In industrialized countries, NTS usually causes acute gastroenteritis, while extraintestinal infection is uncommon (Graham et al., 2000a). In contrast, recent reports from sub-Saharan Africa, including The Gambia, show that NTS was consistently one of the most common causes of paediatric bacteraemia (Hill et al., 2007; Enwere et al., 2006; Kariuki et al., 2006). In the course of a pneumococcal-conjugate vaccine trial (PVT) in rural Gambia, during a period when Haemophilus influenzae type b had been virtually eliminated, Enwere et al. (2006) showed that NTS was the second most common blood culture isolate after Streptococcus pneumoniae in children with invasive bacterial disease. NTS infection is associated with malaria and anaemia, and this often confuses diagnosis and delays appropriate management (Graham et al., 2000b; Berkley et al., 1999). For example, NTS was the most common isolate from blood culture in Ghanaian children who remained febrile after clearance of malarial parasitaemia (Commey et al., 1994). Global emergence of antibiotic resistant NTS has been described (Gebreyes & Thakur, 2005; Lee et al., 2003; Kariuki et al., 2006; Soler et al., 2006). Surveillance of the distribution and antimicrobial susceptibility patterns of NTS serovars, and molecular characterization of NTS isolates from sick children in sub-Saharan Africa are, however, scanty. Surveillance of NTS disease is important not only to establish the current, regional epidemiology of invasive NTS serovars, but also to monitor the emergence and prevalence of antimicrobial resistance, to detect the mutational changes that may occur over time in the serovars allelic profile, and to link these to changes in the pathogenesis of the commonest serovars.
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METHODS
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Study population.
A total of 94 NTS isolates were obtained from 86 children who were investigated as possible cases of invasive bacterial infection during a PVT in rural Gambia (Enwere et al., 2006). However, for unknown reasons, 35 NTS isolates could not be recovered after long-term storage, and hence were excluded from this particular study. Consequently, 59 of the 94 PVT NTS isolates were used in this study. Three additional NTS isolates were collected from children in the study area who were not enrolled in the PVT. Details of how children were enrolled, followed up and investigated have been described previously (Cutts et al., 2005; Enwere et al., 2006). Briefly, blood cultures were obtained from study children admitted to hospital if they had signs of pneumonia, meningitis or septicaemia, and from children managed as outpatients if they had signs of pneumonia and a temperature of 
38 °C. Laboratory diagnosis of NTS was based on isolation of the bacterium from blood and/or cerebro-spinal fluid. Isolates were stored at –70 °C in 15 % glycerol broth before further testing. The study was approved by the Joint Gambia Government and Medical Research Council Ethics Committee, and ethics committees at the London School of Hygiene and Tropical Medicine and the National Institute of Health, USA.
Microbiological methods.
NTS isolates were obtained as described above from patients who were investigated as possible cases of invasive bacterial infection between August 2000 and April 2004. NTS were isolated from blood using an automated blood-culturing system as previously described (Enwere et al., 2006). NTS were identified using biochemical (bioMérieux analytical profile index, API 20 E) and agglutination (Bio-Stat Salmonella polyvalent agglutinating sera ‘O’ and ‘H’) methods. All strains were serotyped with antisera from the Statens Serum Institute. A total of 50 % of the isolates were sent to Statens Serum Institute for confirmation. When a new serovar was discovered this was sent to the Pasteur Institut, Paris, France, for evaluation.
Antimicrobial susceptibility testing.
Antimicrobial susceptibility was determined by both disc diffusion and MIC methods. The following antimicrobial agents were tested by disc diffusion: ampicillin, cotrimoxazole, chloramphenicol, tetracycline, ciprofloxacin, cefotaxime, gentamicin and nalidixic acid (Oxoid). S. Enteritidis ATCC 13076 was used as a control and data were interpreted as susceptible or resistant according to the table in the publication by the NCCLS (2004). MIC of each isolate was tested by Etest strips (AB Biodisk) following the manufacturer's instructions. The following antimicrobial agents were tested by Etest: ampicillin, cotrimoxazole, tetracycline, chloramphenicol, cefotaxime and ciprofloxacin. The control strain S. Enteritidis ATCC 13076 was included in the test.
The Medical Research Council Microbiology Laboratory submits to the external quality assurance programme of the UK National External Quality Assessment Service (http://www.ukneqas.org.uk).
Multilocus sequence typing (MLST).
NTS isolates were streaked on blood agar and incubated at 37 °C for 18 h. A single colony from each isolate was picked, restreaked and incubated at 37 °C for 18 h. Genomic DNA templates were prepared from a loopful of bacteria using a genomic DNA kit (Qiagen) according to the manufacturer's instructions. MLST was performed as described in the Salmonella MLST database (http://web.mpiib-berlin.mpg.de/mlst). Briefly, the seven genes targeted were aroC, dnaN, hemD, hisD, purE, sucA and thrA. Amplifications for all genes were carried out with approximately 0.2 µg DNA template, 250 µM each dNTP, 2.5 mM MgCl2, 25 pmol primers and 1 U Taq polymerase (Qiagen) in a 25 µl reaction mixture. PCR cycling conditions were a 10 min hold at 94 °C, followed by 34 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, and a final extension at 72 °C for 5 min. Aliquots (2 µl) of reaction mixtures were separated by 1.0 % agarose gel electrophoresis, and visualized with ethidium bromide staining and UV illumination, and using a gel documentation system (Gel Doc 2000; Bio-Rad).
PCR products were sent to Macrogen (www.macrogen.com) for purification and DNA sequencing on both strands. Sequences were edited, and complementary sense and antisense fragments were aligned using Laser Gene DNAStar 7.1 software. The sequences were submitted to the MLST database website (http://web.mpiib-berlin.mpg.de/mlst), and assigned an existing or novel allele type number and sequence type (ST) number defined by the database. This multi-micro-organism database defines a novel allele type as a sequence containing one or more nucleotide changes from existing allele sequences. Composite STs are assigned based on the set of allele types derived from each of the seven loci. STs were analysed for relatedness using the eBURST v3 program (http://eburst.mlst.net). Cluster analysis of allelic profiles was performed by using a categorical coefficient and a graphic method called a minimum spanning tree with Bionumerics software (version 4.0; Applied Maths).
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RESULTS AND DISCUSSION
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This is believed to be the first study from The Gambia describing serovar prevalence and molecular characterization of NTS obtained from patients with an invasive infection. The mean age of the patients studied was 18 months (median 22 months, range 4–24 months) and initial clinical diagnoses (made prior to culture results) are shown in Table 1
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Table 1. Initial clinical diagnoses of cases of invasive NTS disease by serovar in 62 Gambian children aged 2–29 months
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Nine different serovars (Table 2) were identified, including a new serovar, namely Salmonella enterica serovar Dingiri. S. Dingiri was isolated from a 6-month-old male with an initial clinical diagnosis of malaria, but a final clinical diagnosis of anaemia and septicaemia. S. Dingiri, which possesses an antigenic formula of 17:z:1,6, was sensitive to ampicillin, cefotaxime, chloramphenicol, ciprofloxacin, cotrimoxazole and tetracycline, but resistant to gentamicin.
The commonest serovar was S. enterica serovar Enteritidis (80.6 %), followed by S. enterica serovar Typhimurium (8 %). Others include S. enterica serovar Offa (S. Offa), S. enterica serovar Virchow (S. Virchow), S. enterica serovar Stanleyville (S. Stanleyville), S. enterica serovar Dublin (S. Dublin), S. enterica serovar Hull (S. Hull), S. enterica serovar Camberene (S. Camberene) and the new serotype S. Dingiri, for each of which one isolate was obtained. Data on the prevalence of NTS in Africa is limited, and different geographical regions harbour different serovars, and different serovars predominate at different periods of time (Galanis et al., 2006). Nevertheless, a World Health Organization survey in all age groups on the global distribution of Salmonella between 2000 and 2002 showed that among human isolates, S. Enteritidis was the most common serovar, accounting for 65 % of all isolates followed by S. Typhimurium at 12 % and S. Newport at 4 % (Galanis et al., 2006). The global prevalence of NTS concurs with our observation in The Gambia within the same time period. However, in contrast to other parts of Africa (Cameroon, Mali, Morocco, Senegal and Tunisia) in 2002, S. Enteritidis and S. Typhimurium were each reported in equal proportion in all age groups (Galanis et al., 2006). These differences in the pattern of NTS infections may be due to ecological (animal reservoirs) or geographical differences in the Africa subregion. Furthermore, there are also different criteria for investigation in different studies – our patients probably included a higher proportion with clinical pneumonia than most other studies, because pneumonia was the main trigger for doing a blood culture in our study. Other studies may have taken cultures from all children with bacteraemia, or gastroenteritis and bacteraemia (Kariuki et al., 2006; Hill et al., 2007).
Invasive NTS in children results in a high mortality, and rapid administration of appropriate antimicrobial therapy can be life-saving. The susceptibility pattern of NTS serovars in this study showed a high proportion susceptible to cefotaxime (100 %), ciprofloxacin (100 %), nalidixic acid (100 %), chloramphenicol (80.6 %) and gentamicin (54.8 %). Susceptibility to ampicillin (26 %), co-trimoxazole (35 %) and tetracycline (37 %) was much lower. It is encouraging that this study found that Gambian isolates of NTS are still largely sensitive to chloramphenicol, which remains a first line treatment for severe pneumonia, septicaemia and meningitis locally and is more affordable than cefotaxime or ciprofloxacin. In Malawi, NTS isolates are more resistant to chloramphenicol in vitro Walsh et al. (2000). In addition, Hadfield et al. (1985) and Lepage et al. (1984) have shown that multiresistant Salmonella are widespread in Africa.
Interestingly, we observed that S. Enteritidis was less likely to be susceptible to commonly used antimicrobials than was S. Typhimurium (Fig. 1), although the number of S. Typhimurium isolates was low, so there is substantial uncertainty around the estimates of resistance for this serovar. The reason for the marked differences in the susceptibility pattern in S. Enteritidis and S. Typhimurium is not clear, but it could be that the plasmid-resistance genes that often confer resistance to multiple classes of antimicrobials are more widely spread in S. Enteritidis than S. Typhimurium, but this will require further investigation.
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Fig. 1. Comparison of susceptibility pattern between S. Enteritidis (black bars) and S. Typhimurium (hatched bars).
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Comparisons were made between disc diffusion, MIC test and MIC agar dilution methods (data not shown) for assessing antimicrobial sensitivity. The results obtained from these methods concur. Our observations show that disc diffusion susceptibility method can be a reliable technique provided that experienced laboratory personnel conduct the test and strictly adhere to the test procedure with inclusion of appropriate controls. In a developing country like The Gambia, the disc diffusion method has advantages as a means of guiding treatment because it is inexpensive, easy to perform and the materials are readily available, in contrast to MIC test, Etest and agar dilution methods, which are very expensive and laborious.
MLST is now the method of choice for the detailed characterization of a number of bacterial pathogens (Aanensen & Spratt, 2005; Sukhnanand et al., 2005; Torpdahl et al., 2005; Urwin & Maiden, 2003). Although it has been reported that MLST of several housekeeping genes provides a satisfactory level of discrimination among diverse Salmonella isolates (Kotetishvili et al., 2002), recent studies suggest that MLST may not be suitable for distinguishing closely related strains within a particular serovar, due to high sequence identity and the slow accumulation of variations in their housekeeping genes (Sukhnanand et al., 2005). Torpdahl et al. (2005) presented MLST data on 25 serotypes of S. enterica using the same 7-gene MLST scheme used in this study, and found that, overall, discrimination was not improved within a serotype compared with PFGE and amplified fragment length polymorphism data. This study confirms that, in general, STs are restricted to the same serovar, especially in the case of S. Enteriditis, and the results from our dataset (Table 1
) and the Salmonella MLST database (http://web.mpiib-berlin.mpg.de/mlst) were concordant. Interestingly, we discovered five novel STs (Table 2) including: (a) an S. Dingiri novel ST (ST338), which shares only two alleles with human S. Javiana ST24, and S. Saintpaul ST126 isolates reported in the USA and S. Oranienburg ST174, found in snakes and lizards in Germany; (b) an S. Stanleyville ST, designated ST339, which is a tri-locus variant with S. Typhimurium ST19, ST98, ST99 and ST153, and S. Thompson ST25; (c) an S. Hull ST340, which shares two alleles with an unknown serovar in the MLST database and with ST202 found in sheep; (d) an S. Offa ST341, which shares three alleles with S. Mbandaka ST206 found in swine in Canada; (e) an S. Camberene ST342, which shares three alleles with S. Senftenberg ST218 found in China.
Furthermore, MLST results showed that S. Typhimurium exhibits two STs, namely ST19 and ST34. ST19 and ST34 are single locus variants at the dnaN locus, and sequence analysis showed that the different alleles at this locus (dnaN 7 and dnaN 19) differ at a single base pair (G to A), suggesting a spontaneous mutation event caused the change in allele type at this locus. As this is believed to be the first report characterizing NTS from developing countries using the seven genes selected for the global MLST Salmonella database housed at the Max-Planck Institut für Infektionsbiologie, Germany (http://web.mpiib-berlin.mpg.de/mlst/), further study using this scheme with a larger number of isolates and additional serovars from Africa is necessary to fully explore the utility of MLST for epidemiological purposes.
The association of NTS infection with a number of clinical conditions has been well documented. In The Gambia, Mabey et al. (1987), found that patients with NTS septicaemia had a significantly higher prevalence of malarial parasitaemia than did patients with other forms of septicaemia (P<001). We observed that 40.3 % (25) of the total NTS cases were associated with an initial clinical diagnosis of malaria, whilst 27.3 % were associated with acute lower respiratory infection and 18 % with septicaemia (Table 1).
Human immunodeficiency virus (HIV) is another important predisposing factor for invasive NTS infections, although the reason for this is not clear. Published studies (Gilks et al., 1990; Nathoo et al., 1996) have demonstrated an association between HIV and bacterial infections, and Rongkavilit et al. (2000) have reported that in the USA and Europe Salmonella bacteraemia is commonly associated with childhood HIV infection. HIV status was not investigated in the children in our study but few are likely to have been HIV positive as the overall prevalence of HIV infection in the adult Gambian population is about 2 % (Schim van der Loeff et al., 2003).
This study has confirmed previous findings from The Gambia (O'Dempsey et al., 1994; Mabey et al., 1987) and from other parts of sub-Saharan Africa that NTS is an important cause of serious illness in young children. However, very little is known about the epidemiology of this infection, for example how the infection is transmitted to young children and whether invasive infections are recently acquired or represent a reactivation of a previously established, asymptomatic infection. The development of new tools to characterize NTS opens the way to more detailed epidemiological studies of this important infection.
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ACKNOWLEDGEMENTS
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We acknowledge the use of the Salmonella MLST database (http://web.mpiib-berlin.mpg.de/mlst), which is housed at Max Planck Institute, Berlin, Germany. The Gambia Pneumococcal Vaccine Trial was funded by grants from: the National Institute of Allergy and Infectious Diseases at the USA National Institutes of Health through contract no. 1-AI-25477; the World Health Organization through contract V23/181/127; the Children's Vaccine Program at PATH; and the USA Agency for International Development.
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REFERENCES
|
|---|
Aanensen, D. M. & Spratt, B. G. (2005). The multilocus sequence typing network: mlst.net. Nucleic Acids Res 33, W728–W733.[Abstract/Free Full Text]
Berkley, J., Mwarumba, S., Bramham, K., Low, B. & Marsh, K. (1999). Bacteraemia complicating severe malaria in children. Trans R Soc Trop Med Hyg 93, 283–286.[CrossRef][Medline]
Berkley, J. A., Lowe, B. S., Mwangi, I., Williams, T., Bauni, E., Mwarumba, S., Ngetsa, C., Slack, M. P., Njenga, S. & other authors (2005). Bacteremia among children admitted to a rural hospital in Kenya. N Engl J Med 352, 39–47.[Abstract/Free Full Text]
Commey, J., Quarm-Goka, B. & Agyepong, I. (1994). Persistent fever in severe malaria in children. Cent Afr J Med 40, 257–260.[Medline]
Cutts, F. T., Zaman, S. M., Enwere, G., Jaffar, S., Levine, O. S., Okoko, J. B., Oluwalana, C., Vaughan, A., Obaro, S. K. & other authors (2005). Efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in The Gambia: randomised, double-blind, placebo-controlled trial. Lancet 365, 1139–1146.[CrossRef][Medline]
Enwere, G., Biney, E., Cheung, Y. B., Zaman, S. M., Okoko, B., Oluwalana, C., Vaughan, A., Greenwood, B., Adegbola, R. & Cutts, F. T. (2006). Epidemiologic and clinical characteristics of community-acquired invasive bacterial infections in children aged 2–29 months in The Gambia. Pediatr Infect Dis J 25, 700–705.[CrossRef][Medline]
Galanis, E., Lo Fo Wong, D. M., Patrick, M. E., Binsztein, N., Cieslik, A., Chalermchikit, T., Aidara-Kane, A., Ellis, A., Angulo, F. J. & Wegener, H. C. (2006). Web-based surveillance and global Salmonella distribution, 2000–2002. Emerg Infect Dis 12, 381–388.[Medline]
Gebreyes, W. A. & Thakur, S. (2005). Multidrug-resistant Salmonella enterica serovar Muenchen from pigs and humans and potential interserovar transfer of antimicrobial resistance. Antimicrob Agents Chemother 49, 503–511.[Abstract/Free Full Text]
Gilks, C. F., Brindle, R. J., Otieno, L. S., Simani, P. M., Newnham, R. S. & Bhatt, S. M. (1990). Life threatening bacteraemia in HIV-1 seropositive adults admitted to hospital in Nairobi, Kenya. Lancet 336, 545–549.[CrossRef][Medline]
Graham, S. M., Molyneux, E. M., Walsh, A. L., Cheesbrough, J. S., Molyneux, M. E. & Hart, A. (2000a). Nontyphoidal Salmonella infection of children in tropical Africa. Pediatr Infect Dis J 19, 1189–1196.[Medline]
Graham, S. M., Walsh, A. L., Molyneoux, E. M., Phiri, A. & Molyneoux, M. E. (2000b). Clinical presentation of non-typhoidal Salmonella bacteraemia Malawian children. Trans R Soc Trop Med Hyg 94, 310–314.[CrossRef][Medline]
Hadfield, T. L., Monson, M. H. & Wachsmuth, I. K. (1985). An outbreak of antibiotic-resistant Salmonella enteritidis in Liberia, West Africa. J Infect Dis 151, 790–795.[Medline]
Hill, P. C., Onyeama, C. O., Ikumapayi, U. N. A., Secka, O., Ameyaw, S., Donkor, S. A., Howie, S. R., Tapgun, M., Corrah, T. & Adegbola, R. A. (2007). Bacteraemia in patients admitted to an urban hospital in West Africa. BMC Infect Dis 7, 2[CrossRef][Medline]
Kariuki, S., Revathi, G., Kariuki, N., Kiiru, J., Mwituria, J. & Hart, C. A. (2006). Characterisation of community acquired non-typhoidal Salmonella from bacteraemia and diarrhoeal infections in children admitted to hospital in Nairobi, Kenya. BMC Microbiol 6, 101[CrossRef][Medline]
Kotetishvili, M., Stine, O. C., Kreger, A., Morris, J. G., Jr & Sulakvelidze, A. (2002). Multilocus sequence typing for characterization of clinical and environmental Salmonella strains. J Clin Microbiol 40, 1626–1635.[Abstract/Free Full Text]
Lee, W. S., Puthucheary, S. D., Parasakthi, N. & Choo, K. E. (2003). Antimicrobial susceptibility and distribution of non-typhoidal Salmonella serovars isolated in Malaysian children. J Trop Pediatr 49, 37–41.[Abstract/Free Full Text]
Lepage, P., Bogaerts, J. & Nsengumuremyi, F. (1984). Severe multiresistant Salmonella typhimurium systemic infections in Central Africa: clinical features and treatment in a paediatric department. J Antimicrob Chemother 14, Suppl. B 153–159.[Abstract/Free Full Text]
Mabey, D. C., Brown, A. & Greenwood, B. M. (1987). Plasmodium falciparum malaria and Salmonella infections in Gambia children. J Infect Dis 155, 1319–1321.[Medline]
Mulholland, E. K. & Adegbola, R. A. (2005). Bacterial infections: a major cause of death among children in Africa. N Engl J Med 352, 75–77.[Free Full Text]
Nathoo, K. J., Chigonde, S., Nhembe, M., Ali, M. H. & Mason, P. R. (1996). Community-acquired bacteraemia in human immunodeficiency virus-infected children in Harare, Zimbabwe. Pediatr Infect Dis J 15, 1092–1097.[CrossRef][Medline]
NCCLS (2004). Performance Standards for Antimicrobial Susceptibility Testing, 14th informational supplement, M100-S14. Wayne, PA: National Committee for Clinical Laboratory Standards.
O'Dempsey, T. J., McArdle, T. F., Lloyd-Evans, N., Baldeh, I., Laurence, B. E., Secka, O. & Greenwood, B. M. (1994). Importance of enteric bacteria as a cause of pneumonia, meningitis and septicemia among children in a rural community in The Gambia, West Africa. Pediatr Infect Dis J 13, 122–128.[Medline]
Rongkavilit, C., Rodriguez, Z. M. & Gomez-Martin, O. (2000). Gram-negative bacillary bacteremia in human immunodeficiency virus type 1-infected children. Pediatr Infect Dis J 19, 122–128.[CrossRef][Medline]
Schim van der Loeff, M. F., Sarge-Njie, R., Ceesay, S., Awasana, A. A., Jaye, P., Sam, O., Jaiteh, K. O., Cubitt, D., Milligan, P. & Whittle, H. C. (2003). Regional differences in HIV trends in The Gambia: results from sentinel surveillance among pregnant women. AIDS 17, 1841–1846.[CrossRef][Medline]
Soler, P., Gonzalez-Sanz, R., Bleda, M. J., Hernandez, G., Echeita, A. & Usera, M. A. (2006). Antimicrobial resistance in non-typhoidal Salmonella from human sources, Spain, 2001–2003. J Antimicrob Chemother 58, 310–314.[Abstract/Free Full Text]
Sukhnanand, S., Alcaine, S., Warnick, L. D., Su, W. L., Hof, J., Craver, M. P., McDonough, P., Boor, K. J. & Wiedmann, M. (2005). DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes. J Clin Microbiol 43, 3688–3698.[Abstract/Free Full Text]
Torpdahl, M., Skov, M. N., Sandvang, D. & Baggesen, D. L. (2005). Genotypic characterization of Salmonella by multilocus sequence typing, pulsed-field gel electrophoresis and amplified fragment length polymorphism. J Microbiol Methods 63, 173–184.[CrossRef][Medline]
Urwin, R. & Maiden, M. C. (2003). Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol 11, 479–487.[CrossRef][Medline]
Walsh, A. L., Phiri, A. J., Graham, S. M., Molyneoux, E. M. & Molyneoux, M. E. (2000). Bacteremia in febrile Malawian children: clinical and microbiological features. Pediatr Infect Dis J 19, 312–318.[CrossRef][Medline]
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INTRODUCTION
METHODS
