Diabetes Care
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published online May 1, 2007
Diabetes Care 30:2051-2056, 2007
DOI: 10.2337/dc07-0461
© 2007 by the American Diabetes Association
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
dc07-0461v1
dc07-0461v2
30/8/2051    most recent
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sotto, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sotto, A.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Pathophysiology/Complications
Original Article

Miniaturized Oligonucleotide Arrays

A new tool for discriminating colonization from infection due to Staphylococcus aureus in diabetic foot ulcers

Albert Sotto, MD, PHD1, Jean-Louis Richard, MD2, Nathalie Jourdan, MD3, Christophe Combescure, PHD4, Nicole Bouziges, MD1,5, Jean-Philippe Lavigne, MD, PHD1,5 on behalf of the Nîmes University Hospital Working Group on the Diabetic Foot (GP30)

1 Institut National de la Santé et de la Recherche Médicale, ESPRI 26, Université de Montpellier 1, UFR de Médecine, Nîmes Cedex 02, France
2 Department of Nutrition and Diabetes, Medical Center, Le Grau du Roi, France
3 Department of Metabolic and Endocrine Diseases, Carémeau University Hospital, Nîmes, France
4 Department of Medical Information, Carémeau University Hospital, NÎmes, France
5 Department of Bacteriology, Carémeau University Hospital, Nîmes, France

Address correspondence and reprint requests to Jean-Philippe Lavigne, Institut National de la Santé et de la Recherche Médicale, ESPRI 26, Université de Montpellier 1, UFR de Médecine, Avenue Kennedy, 30908 Nîmes Cedex 02, France. E-mail: jean.philippe.lavigne{at}chu-nimes.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS--
 CONCLUSIONS--
 References
 
OBJECTIVE—We sought to evaluate the use of oligonucleotide arrays to discriminate colonization from infection due to Staphylococcus aureus in diabetic foot ulcers.

RESEARCH DESIGN AND METHODS—We included diabetic patients hospitalized in a diabetic foot department for an episode of foot ulcer. Only patients who had no antibiotic treatment during the previous 6 months were included. At admission, ulcers were classified on clinical examination, according to the Infectious Diseases Society of America system. Seventy-two patients with a culture positive only for S. aureus as the sole pathogen were included. In individuals with a grade 1 ulcer, a second wound bacterial specimen was obtained 1 month later. Using oligonucleotide arrays, S. aureus resistance and virulence genes were compared between grade 1 and grades 2–4 ulcers.

RESULTS—S. aureus was initially isolated from 22 grade 1 and 50 grade 2–4 ulcers: 35 were methicillin resistant and 37 methicillin sensitive. In 20 grade 1 ulcers (92%), no virulence genes were identified, whereas these genes were present in all but 1 grade 2–4 ulcers. During follow-up, the two grade 1 ulcers that were infected with strains carrying virulence genes rapidly deteriored; the array technology showed unchanged genotype profiles. On the contrary, two grade 1 ulcers healed: the genotype profiles were different from those at inclusion but without appearance of virulence genes.

CONCLUSIONS—The DNA array appears as a promising technique and is easy to perform. Our observational study suggests that it might help distinguish colonized grade 1 from infected grade 2 wounds, predict ulcer outcome, and contribute to a more adequate use of antibiotics.

Abbreviations: IDSA, Infectious Diseases Society of America • MRSA, methicillin-resistant Staphylococcus aureus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESEARCH DESIGN AND METHODS
 RESULTS--
 CONCLUSIONS--
 References
 
Foot ulcers are common in diabetic patients, affecting 15–25% of them during their life. Forty to eighty percent of ulcers eventually become infected (1,2). These infections most often originate as skin ulcers spreading to soft tissue and eventually to bone structures (3). Staphylococcus aureus is by far the most common and most virulent pathogen in diabetic foot infections (4). Because of the dramatically increased risk of amputation due to foot infection in diabetic individuals, early diagnosis and adequate treatment are essential. However, as emphasized by the Infectious Diseases Society of America (IDSA) and the International Working Group on the Diabetic Foot, one of the major concerns in diabetic foot pathology is how to differentiate infection from colonization and, thus, grade 2 (or higher) from grade 1 ulcers according to the IDSA classification system (4,5). As microorganisms are always present on every skin wound, diagnosis of infection should not be based on microbiological findings but on clinical criteria. However, this is often problematic, resulting in misuse of antibiotics (3,6). In turn, inappropriate antibiotic usage contributes to the increasing prevalence of multidrug-resistant bacteria strains, notably methicillin-resistant S. aureus (MRSA) (4,5,7–11). Therefore, assessing strategies to prevent emergence and spread of resistance among organisms is of paramount interest. One major point in this regard includes early differentiation between colonization and infection.

Recently, a miniaturized oligonucleotide array covering the genes encoding resistance determinants, toxins, and species-specific sequences of S. aureus was developed. This rapid and reliable genotyping method that identifies 33 different S. aureus targets was validated under routine conditions (12). The aim of our study was to evaluate the potential of this method for differentiating infection from colonization and predicting grade 1 ulcers.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
RESULTS--
 CONCLUSIONS--
 References
 
Prospective study
Between 1 January 2004 and 30 April 2006, we prospectively enrolled all diabetic patients admitted to our department with any type of foot ulcer. Patients were included if they had never been treated by antibiotics (first episode) or if systemic antibiotic treatment was stopped at least 6 months before the time of onset of the current episode (recurrent wound). Using the IDSA clinical criteria (5) recently validated by Lavery et al. (13), wounds were considered either colonized (grade 1) or infected (grade ≥2). After wound debridement, samples for bacterial culture were obtained by swabbing the wound base, by needle aspiration, or by tissue biopsies and immediately sent to the bacteriology department. To minimize bias, laboratory technicians were kept blind to the clinical data. Only patients with monomicrobial culture positive for S. aureus were retained for inclusion in the study. Patients with grade 1 ulcers did not receive antibiotic treatment but were closely followed up to definitively establish the wound status (infection/colonization) at 1 month after inclusion and until 6 months thereafter. If any worsening was noticed by the patient, they were instructed to come immediately in our outpatient department. If this evolution appeared during the first 3 weeks, the wounds were considered to have a rapid worsening evolution. If the symptoms were observed after 3 weeks, the wounds were considered in a slow worsening evolution. In case of ulcer healing (complete re-epithelialization), a microbiological specimen was obtained 1 month later. Otherwise, a sample was obtained during the worsening phase of the ulcer, and ulcer grade was updated. Ulcer healing was definitively assessed at 6 months after inclusion.

Genus, species, and antibiotic susceptibilities were determined using the Vitek 2 card (BioMérieux, Marcy-l'Etoile, France). Strains were classified as antibiotic sensitive, intermediately resistant, or resistant, according to the recommendations of the Antibiotic Committee of the French Society for Microbiology (14). Sensibility to methicillin was screened by agar diffusion test using cefoxitin (30 µg) disks (BioRad, Marnes La Coquette, France) (14).

Oligonucleotide DNA arrays and genotyping
Each S. aureus strain collected during the study was analyzed in the INSERM laboratory. Genomic DNAs were prepared as previously described (12). A primer elongation reaction was used before hybridization with the DNA array. A 20-µl aliquot of this labeled sample was then transferred into the ArrayTube (ClonDiag, Jena, Germany) and incubated for 60 min at 50°C. The staining reaction was performed according to the manufacturer's instructions. The ArrayTube was then placed into the ATR 04 reading device (Ivagen, Bernis, France). The IconoClust software was used according to the manufacturer for recording and analyzing the arrays. Signal intensity and local background were measured for each probe position. Intensity of local background was subtracted from those of the automatically recognized spots, and averages for all results of a given probe were calculated. Resulting values <0.1 were considered as negative, >0.3 as positive, and between 0.1 and 0.3 as equivocal. The studied virulence genes encoded for the main toxins produced by S. aureus during cutaneous infections included: leukocidins (lukF, lukS, lukS-PV, and lukF-PV), enterotoxins (sea, seb, sec, sek, and seq), exfoliatins (etA and etB), and toxic shock syndrome toxin (tsst). Validation was performed using negative (Staphylococcus epidermidis strains) and positive (S. aureus, where PCRs were developed for every target included in the present study) controls. Validation of mecA genes and virulence genes was obtained by PCR using standard in-house PCR (mecA) and previously published methods (12,16). Finally, arrays results and clinical data were compared and analyzed by two independent experts.

Statistics
For each qualitative variable, comparisons between grade 1 and 2 ulcers and between grade 1 and 2 and grade 3 and 4 ulcers were assessed using the {chi}2 test and Fisher's exact test. A P value ≤0.05 was considered statistically significant. Analysis was performed with SAS software, version 9.1 (Cary, NC).


   RESULTS—
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS--
CONCLUSIONS--
 References
 
From 338 selected patients, 72 were included; in 38 patients the wound was the first episode of ulceration, whereas in 34 it was recurrent. The distribution and characteristics of the included patients are shown in Fig. 1 and Table 1. Twenty-two wounds (30.6%) were classified as grade 1 and followed up for 6 months, 21 ulcers as grade 2 (29.2%), 20 as grade 3 (27.8%), and 9 as grade 4 (12.5%). Thirty-five (48.6%) of the 72 S. aureus we isolated were methicillin resistant (MRSA), and 37 (51.4%) were methicillin sensitive. All recurrent ulcers were positive for MRSA. Only one ulcer appearing as the first episode was positive for MRSA.


Figure 1
View larger version (36K):
[in this window]
[in a new window]

  		Figure 1— Flow of patients through the study and examples of hybridization pictures of grade 1 ulcers during inclusion and follow-up. In grade 1 ulcers, two array profiles were obtained: profile 1, without virulence genes; profile 2, with some virulence genes. During follow-up, if the wound healed, array profiles (profile 3) are different from inclusion profiles (profile 1) without virulence genes. Also, if a wound presented a slow worsening evolution, array profiles (profile 4) are different from inclusion profiles (profile 1) with virulence genes in all of the follow-up cases. On the other hand, if the wound presented a rapid worsening evolution, array profiles (profile 2) are exactly the same in inclusion and follow-up with virulence genes. aGrades according to the International Working Group on the Diabetic Foot and IDSA classifications system (4,5). bIn grade 2, 20 S. aureus (95.2%) had virulence genes and 1 S. aureus (4.8%) had no virulence gene. *Profile identical at initial sample. DFU, diabetic foot ulceration.

 

View this table:
[in this window]
[in a new window]

  		Table 1— Demographic and clinical characteristics of study patients

 
During the follow-up period, 8 grade 1 ulcers healed (36.4%), whereas 14 worsened. Two of 14 had a rapid worsening evolution (≤10 days) and 12 of 14 a slow worsening evolution (≥27 days). Two healing ulcers had samples that remained positive for S. aureus compared with 11 of the 14 nonhealed ulcers. During the whole study, 85 S. aureus were isolated (72 on the initial cultures and 13 during the follow-up period): 48 were methicillin-sensitive S. aureus and 37 MRSA (43.5%).

The prevalence of resistance and virulence determinants are summarized in Table 2. Results of the resistant genes completely agreed with conventional susceptibility data (data not shown). All MRSA isolates were detected by cefoxitin test and were positive for mecA by both PCR and arrays. ermC and ermA macrolide resistance genes were found in 32.9% and 27.1% of the isolates, respectively. The most prevalent aminoglycoside resistance gene was aadD (23.5% of the isolates). The tet, mupR, and dfrA genes were uncommon (<7% of the isolates). No van genes were detected, in agreement with the in vitro susceptibility data. No significant trend in distribution was observed along the different grades, except for the aadD gene, which was more frequently associated with severe grades (2–4) (P = 0.04). A trend was also observed for a higher prevalence of mecA with increasing severity of infection; nevertheless, the difference was statistically significant only for grade 1 compared with grade 4 (P < 0.05).


View this table:
[in this window]
[in a new window]

  		Table 2— Prevalence of resistance determinants among S. aureus strains isolated from diabetic foot ulcers

 
Concerning virulence genes, enterotoxins were detected in 43.5% of the isolates: sea (43.5%) and sec (10.6%) genes were the most prevalent. lukF and lukS leukocidin genes were found in 68.2% and 63.5% of the isolates, respectively. Interestingly, genes encoding Panton-Valentine leukocidin, toxic shock syndrome toxin, and exfoliatin A toxins were found in 1.2, 1.2, and 4.7% of the isolates, respectively. lukF, lukS, sea, and sek were significantly more frequent in grade 2 ulcers compared with grade 1 (P < 0.001).

On admission, S. aureus cultured from 20 of 22 grade 1 ulcers showed no virulence genes, whereas these genes were present in all but one S. aureus isolated from grade 2–4 ulcers. The enterotoxin A gene (sea) was significantly associated with severe grades (3 and 4) (P < 0.001).

At follow-up, S. aureus was isolated from 13 grade 1 ulcers (Table 3). S. aureus from the two healing ulcers were of a different genotype profile compared with the initial sample, only due to difference in resistance genes. However, in both cases, no virulence genes were detected in initial or in follow-up samples. Two of the 11 worsening ulcers showed a rapid deterioration. Both genotype profiles of isolated S. aureus were unchanged. Unlike strains from other grade 1 ulcers, they carried virulence genes. S. aureus from slowly worsening recalcitrant ulcers showed genotype profiles completely different from those at inclusion, with appearance of many virulence genes in every case (Fig. 1).


View this table:
[in this window]
[in a new window]

  		Table 3— Virulence profiles of S. aureus isolated from grade 1 ulcers at initial sampling and follow-up

 

   CONCLUSIONS—
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS--
 CONCLUSIONS--
References
 
Our study suggests that DNA arrays may be a useful tool for rapidly determining S. aureus genotype profiles and differentiating infection from colonization. Over the last few years, different DNA array technologies have been developed, but most systems are very expensive, time consuming, technically demanding, and difficult to adapt to the needs of clinical screening, restricting their use to research laboratories (12,16). In contrast, the miniaturized oligonucleotide array described in this study is relatively easy to perform; the use of tube-integrated arrays (Lab-on Chip system) with nonfluorescent labeling and a rapid hybridization protocol is time saving, easy to use and interpret, and allows large numbers of samples to be analyzed (12). An isolate, once cultured, can be processed within 1 day.

This study confirmed the correlation between results for arrays and PCR as previously demonstrated (12). Nearly one-half of S. aureus we isolated were methicillin resistant. This high-prevalence rate is likely attributable to the high proportion of patients with recurrent ulcers: all were previously infected and had been already treated with antibiotics during a preceding hospitalization, a well-established risk factor for selecting antibiotic-resistant organisms, especially in a diabetic foot clinic (2,4,5,8). However, in grade 1 ulcers, the prevalence of MRSA was limited (29.2%), ruling out the possibility for a clonal transmission of MRSA to explain our results. Finally, an additional interest of the array technology was its ability to identify Panton-Valentine genes; these genes coding for a cytotoxin are thought to be a major threat in severe tissue necrosis (17–21). Interestingly, these genes were present in a strain from a deep ulcer (grade 4).

The main result of our observational study is to show that the microarray technology enables clinicians to distinguish grade 1 from grade 2 ulcers, as the former generally displayed a very low level of virulence genes. Moreover, this tool allows us to predict wound outcome. Indeed, the two grade 1 ulcers with strains carrying virulence determinants had a rapidly worsening course, whereas the strains isolated in the initial and the follow-up samples showed exactly the same genotype profile, suggesting that the isolates were the same and that the wounds were actually infected—not only colonized. According to the genotype profile of S. aureus isolated for grade 1 ulcers, an adapted management of diabetic foot ulcers might be proposed. Furthermore, the array results provide rapid data on S. aureus in vitro susceptibility, notably the presence of the mecA gene, an important therapeutic concern.

One of the main limitations in this study is the small number of grade 1 ulcers we followed-up. Due to the low number of patients consulting for grade 1 ulcer with S. aureus as the sole pathogen and the low level of grade 1 wounds with rapid worsening, a large recruitment would be difficult to obtain. However, a large-scale study must be developed to definitively validate this tool. Moreover, the proposed array could be easily expanded to additional target genes and also adapted for testing other main pathogens isolated in diabetic foot ulceration (Enterobacteriaceae, Pseudomonas spp., or streptococci). Thus, this clinical platform appears suitable for use under routine conditions in a microbiology laboratory.

In conclusion, the increasing prevalence of staphylococci resistance and the small number of new antimicrobial drugs must stimulate the discovery of new solutions in the near future. The miniaturized oligonucleotide arrays may be an interesting tool in management of diabetic foot ulceration, allowing an earlier discrimination of infection from colonization of wounds and a more adequate usage of antibiotic treatment.


   Acknowledgments
 
This work was supported by the Coloplast Foundation, the French Speaking Association for Diabetes and Metabolic Diseases (ALFEDIAM-Aventis grant), the Fondation pour la Recherche Médicale Languedoc-Roussillon-Rouergue, and l'Université Montpellier 1 (Bonus Qualité Recherche).

We thank all of the participants of the Department of Diabetology for help in recruiting patients.

This study was presented at the 26th Interdisciplinary Symposium on Anti-Infectious Chemotherapy (RICAI), Paris, France, December 2006.


   Footnotes
 
Published ahead of print at http://care.diabetesjournals.org on 11 May 2007. DOI: 10.2337/dc07-0461.

A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C Section 1734 solely to indicate this fact.

Received for publication March 8, 2007. Accepted for publication April 24, 2007.


   References
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS--
 CONCLUSIONS--
 References
 

  1. Mayfield JA, Reiber GE, Sanders LJ, Janisse D, Pogach LM: Preventive foot care in people with diabetes. Diabetes Care 21:2161–2177, 1998[Abstract]
  2. Boulton AJ, Vileikyte L, Ragnarson-Tennvall G, Apelqvist J: The global burden of diabetic foot disease. Lancet 336:1719–1724, 2005
  3. Lavery LA, Armstrong DG, Wunderlich RP, Mohler MJ, Wendel CS, Lipsky BA: Risk factors for foot infections in individuals with diabetes. Diabetes Care 29:1288–1293, 2006[Abstract/Free Full Text]
  4. Lipsky BA, Berendt AR, Deery HG, Embil JM, Joseph WS, Karchmer AW, LeFrock JL, Lew DP, Mader JT, Norden C, Tan JS: Infectious Diseases Society of America: diagnosis and treatment of diabetic foot infections. Clin Infect Dis 39:885–910, 2004[Medline]
  5. International Working Group on the Diabetic Foot: International consensus on the diabetic foot [article online], 2003. Available from http://www.diabetic-foot-consensus.com. Accessed 7 March 2007
  6. Xu L, McLennan SV, Lo L, Natfaji A, Bolton T, Liu Y, Twigg SM, Yue DK: Bacterial load predicts healing rate in neuropathic diabetic foot ulcers. Diabetes Care 30:378–380, 2007[Free Full Text]
  7. Game F, Jeffcoate W: MRSA and osteomyelitis of the foot in diabetes. Diabet Med 21:16–19, 2004[Medline]
  8. Dang CN, Prasad YDM, Boulton AJ, Jude EB: Methicillin-resistant Staphylococcus aureus in the diabetic foot clinic: a worsening problem. Diabet Med 20:159–161, 2003[Medline]
  9. Tentolouris N, Petrokkos G, Vallianou N, Zachos C, Daikos GL, Tsapogas P, Markou G, Katsilambros N: Prevalence of methicillin-resistant Staphylococcus aureus in infected and uninfected diabetic foot ulcers. Clin Microbiol Infect 12:186–189, 2006[Medline]
  10. Lipsky BA, Berendt AR: Principles and practice of antibiotic therapy of diabetic foot infections. Diabetes Metab Res Rev 16(Suppl. 1):S42–S46, 2000
  11. Hartemann-Heurtier A, Robert J, Jacqueminet S, Ha Van G, Golmard JL, Jarlier V, Grimaldi A: Diabetic foot ulcers and multidrug-resistant organisms: risk factors and impact. Diabet Med 21:710–715, 2004[Medline]
  12. Monecke S, Ehricht R: Rapid genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolates using miniaturised oligonucleotide arrays. Clin Microbiol Infect 11:825–833, 2005[Medline]
  13. Lavery LA, Armstrong DG, Murdoch DP, Peters EJ, Lipsky BA: Validation of the Infectious Diseases Society of America's diabetic foot infection classification system. Clin Infect Dis 44:562–565, 2007[Medline]
  14. Soussy CJ, Carret G, Cavallo JD, the Antibiotic Susceptibility Testing Committee of the French Society for Microbiology: Antibiotic susceptibility testing [article online], 2006. Available from http://www.sfm.asso.fr. French Society for Microbiology. Accessed 7 March 2007
  15. Lipsky BA: International consensus group on diagnosing and treating the infected diabetic foot: a report from the international consensus on diagnosing and treating the infected diabetic foot. Diabetes Metab Res Rev 20(Suppl. 1):S68–S77, 2004
  16. Heller MJ: DNA microarray technology: devices, systems, and applications. Annu Rev Biomed Eng 4:129–153, 2002[Medline]
  17. Lina G, Piemont Y, Godail-Gamot F, Bes M, Peter MO, Gauduchon V, Vandenesch F, Etienne J: Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 29:1128–1132, 1999[Medline]
  18. Jarraud S, Mougel C, Thioulouse J, Lina G, Meugnier H, Forey F, Nesme X, Etienne J, Vandenesch F: Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect Immun 70:631–641, 2002[Abstract/Free Full Text]
  19. Vandenesch F, Etienne J: How to prevent transmission of MRSA in the open community? Euro Surveill 9:5, 2004[Medline]
  20. Robinson DA, Kearns AM, Holmes A, Morrison D, Grundmann H, Edwards G, O'Brien FG, Tenover FC, McDougal LK, Monk AB, Enright MC: Re-emergence of early pandemic Staphylococcus aureus as a community-acquired meticillin-resistant clone. Lancet 365:1256–1258, 2005[Medline]
  21. Dufour P, Gillet Y, Bes M, Lina G, Vandenesch F, Floret D, Etienne J, Richet H: Community-acquired methicillin-resistant Staphylococcus aureus infections in France: emergence of a single clone that produces Panton-Valentine leukocidin. Clin Infect Dis 35:819–824, 2002[Medline]

Add to CiteULike CiteULike   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
dc07-0461v1
dc07-0461v2
30/8/2051    most recent
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sotto, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sotto, A.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Diabetes Diabetes Care Clinical Diabetes Diabetes Spectrum